Molecular flux rates through critical pathways measured by stable isotope labeling in vivo, as biomarkers of drug action and disease activity

ABSTRACT

The methods described herein enable the evaluation of compounds on subjects to assess their therapeutic efficacy or toxic effects. The target of analysis is the underlying biochemical process or processes (i.e., metabolic process) thought to be involved in disease pathogenesis. Molecular flux rates within the one or more biochemical processes serve as biomarkers and are quantitated and compared with the molecular flux rates (i.e., biomarker) from control subjects (i.e., subjects not exposed to the compounds). Any change in the biomarker in the subject relative to the biomarker in the control subject provides the necessary information to evaluate therapeutic efficacy of an administered drug or a toxic effect and to develop the compound further if desired.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. application Ser.No. 13/781,505, filed Feb. 28, 2013, which is a Continuation applicationof U.S. application Ser. No. 13/215,110 (now U.S. Pat. No. 8,401,800),filed Aug. 22, 2011, which was a Divisional application of U.S.application Ser. No. 11/064,197 (now U.S. Pat. No. 8,005,623), whichclaims priority to U.S. Provisional Application Ser. No. 60/546,580filed on Feb. 20, 2004, and U.S. Provisional Application No. 60/581,028filed on Jun. 17, 2004, all of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The invention relates to methods for measuring changes in biochemicalprocesses that underlie various diseases and disorders. Morespecifically, the invention relates to measuring the molecular fluxrates of these biochemical processes for diagnostic, prognostic, andtherapeutic purposes.

BACKGROUND OF THE INVENTION

It is generally accepted in the fields of biology and medicine that thesigns and symptoms of most diseases (the clinical phenotype) aresecondary consequences of underlying biochemical and molecular processeswhich, in turn, are the fundamental driving forces and etiologic factorsresponsible for the disease. In biochemical terms, the processes thatunderlie most diseases can best be described as molecular fluxes throughcomplex biochemical pathways. These underlying biochemical processes(i.e., the flow of molecules through highly complex, adaptive metabolicpathways or networks) are responsible for the initiation and/orprogression of a disease or disorder from pre-clinical to frank clinicalor morbid stages and are therefore the true targets of contemporarymedical therapeutics (e.g., drug, dietary, behavioral or genetictherapies).

Current drug research and medical diagnostics lack validated,reproducible high-throughput measurement tools for measuring changes inkey biochemical processes in vivo, despite the central importance ofthese processes in driving disease progression. The main explanation forthis gap in the contemporary biochemical repertoire is methodologic:molecular fluxes through complex pathways and networks underlie mostdiseases but effective tools for measuring molecular flux rates arelacking. This state of affairs reflects the fact that measurement ofdynamic processes (flux rates, kinetics) in living organisms requiresdifferent tools and models than measurement of static molecularparameters (e.g., concentration, structure, or composition ofmolecules). Accordingly, the notion of targeting rates of biochemicalprocesses, rather than the physical entities or components that comprisethe biological system of interest (e.g., genes, proteins) per se astargets or biomarkers of drug action or of disease activity, is not onlynew but had previously lacked the technical tools for implementation.

Disclosed herein are methods for testing the effects of compounds,combinations of compounds, or mixtures of compounds (i.e., chemicalentities (whether new or old) drugs (e.g., already-approved drugs orknown drugs), drug leads, or drug candidates, toxic agents, biologicalfactors) on molecular flux rates through metabolic pathways and networksin living systems as biomarkers for drug discovery, development andapproval (DDA), medical diagnosis and prognosis, and toxicology.

SUMMARY OF THE INVENTION

The invention is directed toward analyzing biochemical processes thatare involved in, or are believed to be involved in, the etiology orprogression of a disease or disorder. The biochemical process (i.e., theflow of molecules through a targeted metabolic pathway or network) isthe focus of analysis (as disclosed herein) since it is the underlyingchanges of the biochemical process (i.e., molecular flux rates) that maybe the significant or authentic target for treatment or diagnosticmonitoring of the disease or disorder.

The invention allows for the comparison between the molecular flux rateswithin one or more metabolic pathways of interest measured from cells,tissues, or organisms that have been exposed to one or more compoundsincluding agents (e.g., drugs, drug candidates, or drug leads) to themolecular flux rates from the one or more metabolic pathways of interestmeasured from non-exposed cells, tissues, or organisms. Non-exposedcells, tissues, or organisms may be cells, tissues, or organisms havinga disease or condition of interest but not yet having been exposed toone or more agents (i.e., compounds) or non-exposed cells, tissues, ororganisms may be cells, tissues, or organisms not having the disease orcondition of interest. Differences between the exposed and non-exposedmolecular flux rates are identified and this information is then used todetermine whether the one or more compounds including agents (orcombinations or mixtures thereof) elicit a change in the one or moremetabolic pathways of interest in the exposed cell, tissue, or organism.The one or more compounds including agents may be administered to amammal and the molecular flux rates calculated and evaluated against themolecular flux rates calculated from an unexposed mammal of the samespecies. Alternatively, the molecular flux rates from the same mammalmay be calculated prior to exposure of the one or more compoundsincluding agents and then the molecular flux rates may be calculated inthe same mammal after exposure to the one or more compounds and thencompared. The mammal may be a human.

In another embodiment, the molecular flux rates are measured in one ormore metabolic pathways involved in the molecular pathogenesis of adisease. In a further embodiment, the one or more metabolic pathways arethe cause of the disease or contribute to the initiation, progression,activity, pathologic consequences, symptoms, or severity of the disease.

In another embodiment, the molecular flux rates are measured in one ormore metabolic pathways of interest from a living organism prior to andafter exposure to one or more compounds to evaluate toxicity. Suchcompounds may be chemical entities or agents. In one variation, the oneor more compounds may be industrial or occupational chemicals. Inanother variation, the one or more compounds may be cosmetics. In yetanother variation, the one or more compounds may be food additives. Andin yet another variation, the one or more compounds may be environmentalpollutants. The toxicity of interest may be end-organ toxicity or anyother toxic endpoint.

Alternatively, exposure of one or more compounds or chemical entitiesmay be to one living organism and the molecular flux rates from the oneor more metabolic pathways may be compared to another unexposed livingorganism of the same species to evaluate toxicity. The toxicity ofinterest may be end-organ toxicity or any other toxic endpoint.

In another embodiment, the molecular flux rates two or more metabolicpathways are measured concurrently. In a further embodiment, themolecular flux rates are measured using stable isotope labelingtechniques. The isotope label may include specific heavy isotopes ofelements present in biomolecules, such as ²H, ¹³C, ¹⁵N, ¹⁸O, ³³S, ³⁴S,or may contain other isotopes of elements present in biomolecules suchas ³H, ¹⁴C, ³²P, ³³P, ³⁵S, ¹²⁵I, ¹³¹I. Isotope labeled precursorsinclude, but are not limited to ²H₂O, ¹⁵NH₃, ¹³CO₂, H¹³CO₃, ²H-labeledamino acids, ¹³C-labeled amino acids, ¹⁵N-labeled amino acids,¹⁸O-labeled amino acids, ³⁴S or ³³S-labeled amino acids, ³H₂O,³H-labeled amino acids, and ¹⁴C-labeled amino acids. The stable isotopesubstrate may be chosen from ²H₂O, ²H-glucose, ²H-labeled amino acids,¹³C-labeled amino acids, ²H-labeled organic molecules, ¹³C-labeledorganic molecules, and ¹⁵N-labeled organic molecules labeled water. Thestable isotope substrate may be labeled water. The labeled water may be²H₂O.

Stable isotope-labeled substrates are incorporated into one or moremolecules comprising one or more metabolic pathways of interest. In thismanner, the molecular flux rates can be determined by measuring, overspecific time intervals, isotopic content and/or pattern or rate ofchange of isotopic content and/or pattern in the targeted molecules, forexample by using mass spectrometry, allowing for the determination ofthe molecular flux rates within the one or more metabolic pathways ofinterest, by use of analytic and calculation methods known in the art.

Alternatively, radiolabeled substrates are contemplated for use in thepresent application wherein the radiolabeled substrates are incorporatedinto one or more molecules comprising one or more metabolic pathways ofinterest. In this manner, the molecular flux rates can be determined bymeasuring radiation and/or radioactivity of the targeted molecules ofinterest within the one or more metabolic pathways of interest by usingtechniques known in the art such as scintillation counting. Themolecular flux rates within the one or more metabolic pathways ofinterest are then calculated, using methods known in the art.

The invention is further directed to one or more isotopically perturbedmolecules. The isotopically perturbed molecules may include one or morestable isotopes. The isotopically perturbed molecules are products ofthe labeling methods described herein. The isotopically perturbedmolecules are collected by sampling techniques known in the art and areanalyzed using appropriate analytical tools such as those describedherein.

In yet another embodiment, the isotopically perturbed molecules arelabeled with one or more radioactive isotopes.

In yet another embodiment, one or more kits are provided that includeisotope-labeled precursors and instructions for using them. The kits maycontain stable-isotope labeled precursors or radioactive-labeled isotopeprecursors or both. Stable-isotope labeled precursors andradioactive-labeled isotope precursors may be provided in one kit orthey may be separated and provided in two or more kits. The kits mayfurther include one or more tools for administering the isotope-labeledprecursors. The kits may also include one or more tools for collectingsamples from a subject.

In yet another embodiment, one or more information storage devices areprovided that include data generated from the methods of the presentinvention. The data may be analyzed, partially analyzed, or unanalyzed.The data may be imprinted onto paper, plastic, magnetic, optical, orother medium for storage and display.

The application is further directed to one or more compounds identifiedand at least partially characterized by the methods of the presentinvention.

The present application is further directed to a method for evaluatingthe action of one or more compounds on a molecular flux rate through acritical pathway as an authentic biomarker of disease, wherein themethod includes: a) exposing a living system to one or more compounds;b) administering an isotope-labeled substrate to the living system for aperiod of time sufficient for the isotope-labeled substrate to enterinto one or more metabolic pathways of interest and thereby enter intoand label at least one targeted molecule of interest within the one ormore metabolic pathways of interest in the living system; c) obtainingone or more samples from the living system, wherein the one or moresamples include at least one isotope-labeled targeted molecule ofinterest; d) measuring the content, rate of incorporation and/or patternor rate of change in content and/or pattern of isotope labeling of theat least one targeted molecule of interest; e) calculating molecularflux rates in the one or more metabolic pathways of interest based onthe content and/or pattern or rate of change of content and/or patternof isotopic labeling in the at least one targeted molecule of interest;f) measuring the molecular flux rates in the one or more metabolicpathways of interest according to steps b) through e) in at least oneliving system not exposed to the one or more compounds as provided bystep a); and g) comparing the molecular flux rates in the one or moremetabolic pathways of interest in the living system administered the oneor more compounds to the molecular flux rates in the one or moremetabolic pathways of interest in the living system not administered theone or more compounds to evaluate the action of the one or morecompounds on the molecular flux rates. An authentic biomarker is theflow of molecules through a targeted metabolic pathway that is involvedin the progression of a disease or disorder.

The molecular flux rates in the one or more metabolic pathways ofinterest may be relevant to an underlying molecular pathogenesis, orcausation of, one or more diseases. Further, the molecular flux rates inthe one or more metabolic pathways of interest may contribute to theinitiation, progression, severity, pathology, aggressiveness, grade,activity, disability, mortality, morbidity, disease sub-classificationor other underlying pathogenic or pathologic feature of the one or morediseases. Further, the molecular flux rates in the metabolic pathways ofinterest may contribute to the prognosis, survival, morbidity,mortality, stage, therapeutic response, symptomology, disability orother clinical factor of the one or more diseases.

In one format of the method, the molecular flux rates of two or moremetabolic pathways of interest are measured concurrently. In anotherformat of the invention, the one or more samples may be collected atknown times or intervals after administration or contacting the livingsystem to the isotope-labeled substrate and after exposing the livingsystem to the one or more compounds.

The concurrent measurement of the molecular flux rates from themetabolic pathways of interest may be achieved by use of stable isotopiclabeling techniques. The isotope label used may be stable (i.e.,non-radioactive) isotope. The stable isotope may be isotope-labeledwater, e.g. ²H₂O.

In one format, the concurrent measurement of the molecular flux ratesfrom the metabolic pathways of interest may be achieved by use ofradioisotope labeling techniques.

In another format, the one or more compounds may be an already-approveddrug, e.g., a Federal Food and Drug Administration-approved drug or adrug approved by a similar agency outside the United States. In oneformat, the already-approved drug is selected randomly. In anotherformat, the already-approved drug may be selected on the basis of aspecific biochemical rationale or hypothesis concerning a hypothesizedrole in the molecular pathogenesis of one or more diseases. In anotherformat, the one or more compounds is a chemical entity (whether new orold) or a biological factor. The already-approved drug may be chosenfrom statins, glitazones, COX-2 inhibitors, NSAIDS, β-blockers, calciumchannel blockers, ACE inhibitors, antibiotics, antiviral agents,hypolipidemic agents, antihypertensives, anti-inflammatory agents,antidepressants, anxiolytics, anti-psychotics, sedatives, analgesics,antihistamines, oral hypoglycemic agents, antispasmodics,antineoplastics, cancer chemotherapeutic agents, sex steroids, pituitaryhormones, cytokines, chemokines, appetite suppressant agents,thyromimetics, anti-seizure agents, sympathomimetics, sulfa drugs,biguanides, and other classes of agents.

In one format of the invention, one or more animal models of disease areused for evaluating the actions on molecular flux rates in one or moremetabolic pathways potentially related to disease in living systems. Theone or more animal models of disease may be chosen from Alzheimer'sdisease, heart failure, renal disease, diabetic nephropathy,osteoporosis, hepatic fibrosis, cirrhosis, hepatocellular necrosis,pulmonary fibrosis, scleroderma, renal fibrosis, multiple sclerosis,arteriosclerosis, osteoarthritis, rheumatoid arthritis, psoriasis, skinphotoaging, skin rashes, breast cancer, prostate cancer, colon cancer,pancreatic cancer, lung cancer, acquired immunodeficiency syndrome,immune defects, multiple myeloma, chronic lymphocytic leukemia, chronicmyelocytic leukemia, diabetes, diabetic complications, insulinresistance, obesity, lipodystrophy, muscle wasting, frailty,deconditioning, angiogenesis, hyperlipidemia, infertility, viral orbacterial infections, auto-immune disorders, and immune flares.

In one format of the invention, the one or more metabolic pathways ofinterest are measured in response to a specific dose or a range of dosesof the one or more compounds.

The one or more metabolic pathways of interest may be chosen fromhepatocyte proliferation and destruction, total liver cell proliferationand destruction, renal tubular cell turnover, lymphocyte turnover,spermatocyte turnover, protein synthesis and breakdown in muscle andheart, liver collagen synthesis and breakdown, myelin synthesis andbreakdown in brain or peripheral nerves, breast epithelial cellproliferation, colon epithelial cell proliferation, prostate epithelialcell proliferation, ovarian epithelial cell proliferation, endometrialcell proliferation, bronchial epithelial cell proliferation, pancreaticepithelial cell proliferation, keratin synthesis in skin, keratinocyteproliferation, immunoglobulin synthesis, synthesis and breakdown ofmitochondrial DNA, synthesis and breakdown of mitochondrialphospholipids, synthesis and breakdown of mitochondrial proteins,synthesis and breakdown of adipose lipids, and synthesis and breakdownof adipose cells.

In yet another format of the invention, the already-approved drug isscreened for actions on multiple biochemical processes concurrently.

In one format, the living system is chosen from prokaryotic cells,eukaryotic cells, cell lines, cell cultures, isolated tissuepreparations, rabbits, dogs, mice, rats, guinea pigs, pigs non-humanprimates, and humans.

In another format, the isotope labeled substrate is chosen from ²H₂O,²H-glucose, ²H-labeled amino acids, ²H-labeled organic molecules,¹³C-labeled organic molecules, ¹³CO₂, ¹⁵N-labeled organic molecules,³H₂O, ³H-labeled glucose, ³H-labeled amino acids, ³H-labeled organicmolecules, ¹⁴C-labeled organic molecules and ¹⁴CO₂.

The one or more compounds may be administered according to establishedor hypothesized dose ranges that have the potential for biologicalactivity in the living system.

In another format of the invention, combinations of two or morecompounds are exposed to the living system. In this format, synergistic,complementary, or antagonistic actions of combinations of compounds onmolecular flux rates through the one or more metabolic pathways aredetermined by comparing the molecular flux rates in the living systemsexposed to the combination of compounds to the molecular flux rates inthe living systems exposed to a single compound alone or not exposed toany of the compounds being tested. In one format, the combinations ofcompounds are selected randomly. The combinations of compounds may beselected on the basis of a specific biochemical rationale or hypothesisconcerning a hypothesized role of one or more of the compounds in themolecular pathogenesis of the one or more diseases.

The present invention is further directed to a method for evaluating anaction on a molecular flux rate through a critical pathway as anauthentic biomarker of toxicity, the method including: a) exposing aliving system to one or more compounds; b) administering anisotope-labeled substrate to a living system for a period of timesufficient for the isotope-labeled substrate to enter into one or moremetabolic pathways of interest and thereby enter into and label one ormore targeted molecules of interest within the one or more metabolicpathways of interest in the living system wherein the one or moremetabolic pathways of interest are related to one or more toxic effects;c) obtaining one or more samples from the living system, wherein the oneor more samples include one or more isotope-labeled targeted moleculesof interest; d) measuring the content, rate of incorporation and/orpattern or rate of change in content and/or pattern of isotope labelingof the targeted molecule or molecules of interest; e) calculatingmolecular flux rates in the one or more metabolic pathways of interestbased on the content and/or pattern or rate of change of content and/orpattern of isotopic labeling in the molecule or molecules of interest;f) measuring the molecular flux rates in the one or more metabolicpathways of interest according to steps b) through e) in a living systemor systems not administered the one or more compounds; and g) comparingthe molecular flux rates in the one or more metabolic pathways ofinterest in the living system administered the one or more compounds tothe molecular flux rates in the one or more metabolic pathways in theliving system or systems not administered the one or more compounds.

The one or more toxic actions may include at least one end-organtoxicity. The end-organ toxicity may be chosen from hepatocyteproliferation and destruction, total liver cell proliferation anddestruction, renal tubular cell turnover, lymphocyte turnover,spermatocyte turnover, protein synthesis and breakdown in muscle andheart, liver collagen synthesis and breakdown, myelin synthesis andbreakdown in brain or peripheral nerves, breast epithelial cellproliferation, colon epithelial cell proliferation, prostate epithelialcell proliferation, ovarian epithelial cell proliferation, endometrialcell proliferation, bronchial epithelial cell proliferation, pancreaticepithelial cell proliferation, keratin synthesis in skin, keratinocyteproliferation, immunoglobulin synthesis, synthesis and breakdown ofmitochondrial DNA, synthesis and breakdown of mitochondrialphospholipids, synthesis and breakdown of mitochondrial proteins,synthesis and breakdown of adipose lipids, and synthesis and breakdownof adipose cells.

The one or more metabolic pathways of interest related to end-organtoxicity may be measured in response to a specific dose or a range ofdoses of the one or more compounds of interest.

The living system may be chosen from prokaryotic cells, eukaryoticcells, cell lines, cell cultures, isolated tissue preparations, rabbits,dogs, mice, rats, guinea pigs, pigs, and non-human primates. Such toxiceffects can be analyzed on isolated human cells or tissue preparationsbut are not performed on humans in vivo. The living system may beexposed to combinations of two or more compounds. Synergistic,complementary, or antagonistic actions of combinations of compounds onmolecular flux rates through the one or more metabolic pathways ofinterest may be determined by comparing the molecular flux rates in theliving systems exposed to the combination of compounds to the molecularflux rates in the living systems exposed to a single compound alone ornot exposed to any of said one or more compounds being tested.

The information generated using the methods of the invention may bestored in an information storage device. The device may be a printedreport. The medium in which the report is printed on may be chosen frompaper, plastic, and microfiche. The device may be a computer disc. Thedisc may be chosen from a compact disc, a digital video disc, an opticaldisc, and a magnetic disc. The device may also be a computer.

The present application is further directed to an isotopically-perturbedmolecule generated by the methods of the invention. The molecule may bechosen from protein, lipid, nucleic acid, glycosaminoglycan,proteoglycan, porphyrin, and carbohydrate molecules. In one format theisotopically perturbed molecule is myelin, amyloid-β, deoxyribonucleicacid, ribonucleic acid, collagen or a triglyceride.

The present application is further directed to a kit for determiningscreening of one or more compounds for actions on molecular flux ratesin one or more metabolic pathways potentially related to disease in asubject, including: a) one or more isotope-labeled precursors and b)instructions for use of the kit. The kit may further include a tool foradministration of precursor molecules or an instrument for collecting asample from the subject.

The present application is further directed to a kit for screening ofone or more compounds for actions on molecular flux rates in one or moremetabolic pathways potentially related to one or more toxic effects in asubject, including: a) one or more isotope-labeled precursors, and b)instructions for use of the kit. The kit may further include a tool foradministration of precursor molecules or an instrument for collecting asample from the subject.

The methods of the application may further include the step ofmanufacturing one or more compounds at least partially identified by themethods of the invention. The methods of the invention may furtherinclude the step of developing one or more compounds at least partiallyidentified by the methods of the invention.

The present application is further directed to a method including:measuring a molecular flux rate of an authentic biomarker of interestusing an isotope; comparing the results of step a) with the molecularflux rate of the authentic biomarker of interest in the presence of acompound of interest; if the compound of interest changes a molecularflux rate of interest, the compound is then further developed.

The present application is further directed to a method for monitoringor diagnosing a clinical or medical disease or condition, the methodincluding: a) administering an isotope-labeled substrate to a livingsystem for a period of time sufficient for the isotope-labeled substrateto enter into one or more metabolic pathways of interest and therebyenter into and label at least one targeted molecule of interest withinthe one or more metabolic pathways of interest in the living system; b)obtaining one or more samples from the living system, wherein the one ormore samples include at least one isotope-labeled targeted molecule ofinterest; c) measuring the content, rate of incorporation and/or patternor rate of change in content and/or pattern of isotope labeling of theat least one targeted molecule of interest; d) calculating molecularflux rates in the one or more metabolic pathways/biomarker of interestbased on the content and/or pattern or rate of change of content and/orpattern of isotopic labeling in the at least one targeted molecule ofinterest to monitor or diagnose the clinical or medical disease orcondition.

Table 1 depicts examples of authentic biomarkers, the related clinicalor medical diseases or conditions and the molecule of interest to bedetected using the methods of the application. Taking into account Table1, the present application is further directed to a method formonitoring or diagnosing a clinical or medical disease or condition, themethod including: a) administering an isotope-labeled substrate to theliving system for a period of time sufficient for the isotope-labeledsubstrate to enter into one or more metabolic pathways of interest andthereby enter into and label at least one targeted molecule of interestwithin the one or more metabolic pathways of interest in the livingsystem; b) obtaining one or more samples from the living system, whereinthe one or more samples include at least one isotope-labeled targetedmolecule of interest; c) measuring the content, rate of incorporationand/or pattern or rate of change in content and/or pattern of isotopelabeling of the at least one targeted molecule of interest; d)calculating molecular flux rates in the one or more metabolicpathways/biomarker of interest based on the content and/or pattern orrate of change of content and/or pattern of isotopic labeling in the atleast one targeted molecule of interest to monitor or diagnose theclinical or medical disease or condition. In another format, one or morecompounds are administered to the living system before or after thedetermination of the molecular flux rates of the one or more metabolicpathways of interest in the living system in order to evaluate theaction of the one or more compounds on the biomarker as a predictor ofan effect of the compound on the clinical or medical disease orcondition.

Various clinical or medical diseases or conditions can be diagnosed ormonitored using the methods of the invention as depicted in Table 1.Each of the clinical or medical diseases or conditions explained in moredetail below can be monitored using the methods of the invention beforeand after the administration of one or more compounds to evaluate theaction of the one or more compounds as a potential treatment, diagnosticor causative agent.

For example, obesity, lipoatrophy, fat distribution, orhyperplasia-hypertrophy can be monitored or diagnosed by measuring ordetecting adipose triglyceride dynamics. In this method, the targetedmolecule of interest is triglyceride glycerol or one or more fattyacids. As such, the present application is further directed to a methodfor monitoring or diagnosing obesity; lipoatrophy; fat distribution orhyperplasia-hypertrophy in a living system, the method including: a)administering an isotope-labeled substrate to a living system for aperiod of time sufficient for the isotope-labeled substrate to enterinto the adipose triglyceride metabolic pathway and thereby enter intoand label at least one triglyceride glycerol or fatty acid within theadipose triglyceride metabolic pathway in the living system; b)obtaining one or more samples from the living system, wherein the one ormore samples include at least one isotope-labeled triglyceride glycerolor fatty acid; c) measuring the content, rate of incorporation and/orpattern or rate of change in content and/or pattern of isotope labelingof the triglyceride glycerol or fatty acid; d) calculating molecularflux rates in the adipose triglyceride metabolic pathway based on thecontent and/or pattern or rate of change of content and/or pattern ofisotopic labeling in the triglyceride glycerol or fatty acid to monitoror diagnose obesity; lipoatrophy; fat distribution and/orhyperplasia-hypertrophy.

Hyperplasia-hypertrophy can be monitored or diagnosed by measuring ordetecting adipocyte dynamics. In this method, the targeted molecule ofinterest is DNA isolated from adipocytes. In this format, the presentapplication is further directed to a method for monitoring or diagnosinghyperplasia-hypertrophy in a living system, the method including: a)administering an isotope-labeled substrate to the living system for aperiod of time sufficient for the isotope-labeled substrate to enterinto the adipose metabolic pathway and thereby enter into and label atleast one DNA molecule isolated from adipocytes within the adiposemetabolic pathway in the living system; b) obtaining one or more samplesfrom the living system, wherein the one or more samples include at leastone isotope-labeled DNA molecule isolated from adipocytes; c) measuringthe content, rate of incorporation and/or pattern or rate of change incontent and/or pattern of isotope labeling of the DNA isolated fromadipocytes; d) calculating molecular flux rates in the adipose metabolicpathway based on the content and/or pattern or rate of change of contentand/or pattern of isotopic labeling in the DNA isolated from adipocytesto monitor or diagnose hyperplasia-hypertrophy.

Unfitness, cardiovascular disease risk, autotoxicity drugs,deconditioning or frailty can be monitored or diagnosed by measuring ordetecting muscle mitochondrial DNA or phospholipid dynamics. In thismethod, the targeted molecules of interest are DNA from musclemitochondria or phospholipids from muscle mitochondria. In this format,the present application is further directed to a method for monitoringor diagnosing unfitness, cardiovascular disease risk, autotoxicitydrugs, deconditioning or frailty in a living system, the methodincluding: a) administering an isotope-labeled substrate to the livingsystem for a period of time sufficient for the isotope-labeled substrateto enter into the muscle mitochondrial DNA or phospholipid metabolicpathway and thereby enter into and label at least one DNA molecule frommuscle mitochondria or one phospholipid from muscle mitochondria withinthe muscle mitochondrial DNA or phospholipid metabolic pathway in theliving system; b) obtaining one or more samples from the living system,wherein the one or more samples include at least one isotope-labeled DNAmolecule from muscle mitochondria or one phospholipid from musclemitochondria; c) measuring the content, rate of incorporation and/orpattern or rate of change in content and/or pattern of isotope labelingof the DNA from muscle mitochondria or phospholipids from musclemitochondria; d) calculating molecular flux rates in the musclemitochondrial DNA or phospholipid metabolic pathway based on the contentand/or pattern or rate of change of content and/or pattern of isotopiclabeling in the DNA from muscle mitochondria or phospholipids frommuscle mitochondria to monitor or diagnose unfitness, cardiovasculardisease risk, autotoxicity drugs, deconditioning or frailty.

Frailty, wasting or dystrophies can be monitored or diagnosed bymeasuring or detecting muscle protein dynamics. In this method, thetargeted molecule of interest is protein derived from muscle. In thisformat, the present application is further directed to a method formonitoring or diagnosing frailty, wasting or dystrophies in a livingsystem, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the muscle protein metabolicpathway and thereby enter into and label at least one protein derivedfrom muscle within the muscle protein metabolic pathway in the livingsystem; b) obtaining one or more samples from the living system, whereinthe one or more samples include at least one isotope-labeled proteinderived from muscle; c) measuring the content, rate of incorporationand/or pattern or rate of change in content and/or pattern of isotopelabeling of the protein derived from muscle; d) calculating molecularflux rates in the muscle protein metabolic pathway based on the contentand/or pattern or rate of change of content and/or pattern of isotopiclabeling in the protein derived from muscle to monitor or diagnosefrailty, wasting or dystrophies.

Atherosclerosis or risk of diabetes mellitus can be monitored ordiagnosed by measuring or detecting dynamics of adipose lipolysis. Inthis method, the targeted molecule of interest is triglyceride glycerolor one or more fatty acids. In this format, the present application isfurther directed to a method for monitoring or diagnosingatherosclerosis or assessing the risk of diabetes mellitus in a livingsystem, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the adipose lipolysis pathwayand thereby enter into and label at least one triglyceride glycerol orfatty acid within the adipose lipolysis pathway in the living system; b)obtaining one or more samples from the living system, wherein the one ormore samples include at least one isotope-labeled triglyceride glycerolor fatty acid; c) measuring the content, rate of incorporation and/orpattern or rate of change in content and/or pattern of isotope labelingof the triglyceride glycerol or fatty acid; d) calculating molecularflux rates in the adipose lipolysis pathway based on the content and/orpattern or rate of change of content and/or pattern of isotopic labelingin the triglyceride glycerol or fatty acid to monitor or diagnoseatherosclerosis or assess the risk of diabetes mellitus.

Carbohydrate overfeeding, anabolic block, impaired fat oxidation orenergy balance can be monitored or diagnosed by measuring or detectingthe dynamics of adipose or hepatic de novo lipogenesis. In this method,the targeted molecule of interest is one or more fatty acids. In thisformat, the present application is further directed to a method formonitoring or diagnosing carbohydrate overfeeding, anabolic block,impaired fat oxidation or energy balance in a living system, the methodincluding: a) administering an isotope-labeled substrate to the livingsystem for a period of time sufficient for the isotope-labeled substrateto enter into the adipose metabolic pathway or hepatic de novolipogenesis pathway and thereby enter into and label at least one ormore fatty acids within the adipose metabolic pathway or hepatic de novolipogenesis pathway in the living system; b) obtaining one or moresamples from the living system, wherein the one or more samples includeat least one isotope-labeled fatty acid; c) measuring the content, rateof incorporation and/or pattern or rate of change in content and/orpattern of isotope labeling of the fatty acid; d) calculating molecularflux rates in the adipose metabolic pathway or hepatic de novolipogenesis pathway based on the content and/or pattern or rate ofchange of content and/or pattern of isotopic labeling in the fatty acidto monitor or diagnose carbohydrate overfeeding, anabolic block,impaired fat oxidation or energy balance.

Insulin resistance, impaired glucose tolerance or diabetes mellitus riskcan be monitored or diagnosed by measuring or detecting the dynamics ofglycolysis. In this method, the targeted molecule of interest is water.In this format, the present application is further directed to a methodfor monitoring or diagnosing insulin resistance, impaired glucosetolerance or diabetes mellitus risk in a living system, the methodincluding: a) administering an isotope-labeled substrate to the livingsystem for a period of time sufficient for the isotope-labeled substrateto enter into glycolysis and thereby enter into and label at least onewater molecule within the glycolysis pathway in the living system; b)obtaining one or more samples from the living system, wherein the one ormore samples include at least one isotope-labeled water molecule; c)measuring the content, rate of incorporation and/or pattern or rate ofchange in content and/or pattern of isotope labeling of the water; d)calculating molecular flux rates in the glycolysis pathway based on thecontent and/or pattern or rate of change of content and/or pattern ofisotopic labeling in the water to monitor or diagnose insulinresistance, impaired glucose tolerance or diabetes mellitus risk.

Obesity risk, hypometabolism or hypermetabolism, or response tocompounds or therapeutics can be monitored or diagnosed by measuring ordetecting the dynamics of metabolic H₂O or CO₂ production. In thismethod, the targeted molecule of interest is water or CO₂. In thisformat, the present application is further directed to a method formonitoring or diagnosing obesity risk, hypo or hypermetabolism, orresponse to compounds or therapeutics in a living system, the methodincluding: a) administering an isotope-labeled substrate to the livingsystem for a period of time sufficient for the isotope-labeled substrateto enter into the metabolic H₂O or CO₂ production pathway and therebyenter into and label at least one water molecule or one CO₂ moleculewithin the metabolic H₂O or CO₂ production pathway in the living system;b) obtaining one or more samples from the living system, wherein the oneor more samples include at least one isotope-labeled water molecule; c)measuring the content, rate of incorporation and/or pattern or rate ofchange in content and/or pattern of isotope labeling of the water; d)calculating molecular flux rates in the metabolic H₂O or CO₂ productionpathway based on the content and/or pattern or rate of change of contentand/or pattern of isotopic labeling in the water to monitor or diagnoseobesity risk, hypometabolism or hypermetabolism, or response tocompounds or therapeutics.

Obesity risk or insulin resistance can be monitored or diagnosed bymeasuring or detecting the dynamics of fatty acid oxidation. In thismethod, the targeted molecule of interest is water. In this format, thepresent application is further directed to a method for monitoring ordiagnosing obesity risk or insulin resistance in a living system, themethod including: a) administering an isotope-labeled substrate to theliving system for a period of time sufficient for the isotope-labeledsubstrate to enter into the fatty acid oxidation pathway and therebyenter into and label at least one water molecule within the fatty acidoxidation pathway in the living system; b) obtaining one or more samplesfrom the living system, wherein the one or more samples include at leastone isotope-labeled water molecule; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the water; d) calculating molecular flux rates inthe fatty acid oxidation pathway based on the content and/or pattern orrate of change of content and/or pattern of isotopic labeling in thewater to monitor or diagnose obesity risk or insulin resistance.

Hepatic insulin resistance, hypometabolism or hypermetabolism ortreatment thereof can be monitored or diagnosed by measuring ordetecting the dynamics of hepatic glucose production. In this method,the targeted molecule of interest is glucose. In this format, thepresent application is further directed to a method for monitoring ordiagnosing hepatic insulin resistance or hypo or hypermetabolism in aliving system, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the hepatic glucose productionpathway and thereby enter into and label at least one glucose moleculewithin the hepatic glucose production pathway in the living system; b)obtaining one or more samples from the living system, wherein the one ormore samples include at least one isotope-labeled glucose molecule; c)measuring the content, rate of incorporation and/or pattern or rate ofchange in content and/or pattern of isotope labeling of the glucose; d)calculating molecular flux rates in the hepatic glucose productionpathway based on the content and/or pattern or rate of change of contentand/or pattern of isotopic labeling in the glucose to monitor ordiagnose hepatic insulin resistance, hypometabolism or hypermetabolismor treatment thereof.

Hepatic steatosis (including tumors and cirrhosis) or treatment thereofcan be monitored or diagnosed by measuring or detecting the dynamics ofhepatic triglyceride synthesis. In this method, the targeted molecule ofinterest is triglyceride glycerol or one or more fatty acids. In thisformat, the present application is further directed to a method formonitoring or diagnosing hepatic steatosis in a living system, themethod including: a) administering an isotope-labeled substrate to theliving system for a period of time sufficient for the isotope-labeledsubstrate to enter into the hepatic triglyceride synthesis pathway andthereby enter into and label at least one triglyceride glycerol or fattyacid within the hepatic triglyceride synthesis pathway in the livingsystem; b) obtaining one or more samples from the living system, whereinthe one or more samples include at least one isotope-labeledtriglyceride glycerol or fatty acid; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the triglyceride glycerol or fatty acid; d)calculating molecular flux rates in the hepatic triglyceride synthesispathway based on the content and/or pattern or rate of change of contentand/or pattern of isotopic labeling in the triglyceride glycerol orfatty acid to monitor or diagnose hepatic steatosis.

Pancreatic burden, pancreatic reserve or diabetes mellitus risk ortreatment thereof can be monitored or diagnosed by measuring ordetecting β-Cell DNA dynamics. In this method, the targeted molecule ofinterest is DNA derived from pancreatic beta cells. In this format, thepresent application is further directed to a method for monitoring ordiagnosing pancreatic burden, pancreatic reserve or diabetes mellitusrisk in a living system, the method including: a) administering anisotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into the β-CellDNA and thereby enter into and label at least one DNA molecule derivedfrom pancreatic beta cells within the β-Cell DNA in the living system;b) obtaining one or more samples from the living system, wherein the oneor more samples include at least one isotope-labeled DNA moleculederived from pancreatic beta cells; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the DNA derived from pancreatic beta cells; d)calculating molecular flux rates in the β-Cell DNA based on the contentand/or pattern or rate of change of content and/or pattern of isotopiclabeling in the DNA derived from pancreatic beta cells to monitor ordiagnose pancreatic burden, pancreatic reserve or diabetes mellitus riskor treatment thereof.

Pancreatic burden or pancreatic reserve or treatment thereof can bemonitored or diagnosed by measuring or detecting insulin dynamics. Inthis method, the targeted molecule of interest is insulin. In thisformat, the present application is further directed to a method formonitoring or diagnosing pancreatic burden/pancreatic reserve in aliving system, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the insulin metabolic pathwayand thereby enter into and label at least one insulin molecule withinthe insulin metabolic pathway in the living system; b) obtaining one ormore samples from the living system, wherein the one or more samplesinclude at least one isotope-labeled insulin molecule; c) measuring thecontent, rate of incorporation and/or pattern or rate of change incontent and/or pattern of isotope labeling of the insulin; d)calculating molecular flux rates in the insulin metabolic pathway basedon the content and/or pattern or rate of change of content and/orpattern of isotopic labeling in the insulin to monitor or diagnosepancreatic burden and/or pancreatic reserve.

Diabetes mellitus complications or treatment thereof can be monitored ordiagnosed by measuring or detecting advanced glycation end productdynamics or advanced glycation end product glycosylation dynamics. Inthis method, the targeted molecules of interest are advanced glycationend products. In this format, the present application is furtherdirected to a method for monitoring or diagnosing Diabetes mellituscomplications in a living system, the method including: a) administeringan isotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into the advancedglycation end product pathway; advanced glycation endproductglycosylation pathway and thereby enter into and label at least oneadvanced glycation end product within the advanced glycation end productpathway or advanced glycation end product glycosylation pathway in theliving system; b) obtaining one or more samples from the living system,wherein the one or more samples include at least one isotope-labeledadvanced glycation end product; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the advanced glycation end products; d)calculating molecular flux rates in the glycation end product pathway oradvanced glycation end product glycosylation pathway based on thecontent and/or pattern or rate of change of content and/or pattern ofisotopic labeling in the advanced glycation end products to monitor ordiagnose Diabetes mellitus complications or treatment thereof.

Caloric restriction/longevity regimens can be monitored or diagnosed bymeasuring or detecting keratinocyte or mammary epithelial cell dynamics.In this method, the targeted molecule of interest is DNA derived fromkeratinocytes or mammary epithelial cells. In this format, the presentapplication is further directed to a method for monitoring or diagnosingcaloric restriction/longevity regimens in a living system, the methodincluding: a) administering an isotope-labeled substrate to the livingsystem for a period of time sufficient for the isotope-labeled substrateto enter into the keratinocyte or mammary epithelial cell productionpathway and thereby enter into and label at least one DNA moleculederived from keratinocytes or mammary epithelial cells within thekeratinocyte or mammary epithelial cell production pathway in the livingsystem; b) obtaining one or more samples from the living system, whereinthe one or more samples include at least one isotope-labeled DNAmolecule derived from keratinocytes or mammary epithelial cells; c)measuring the content, rate of incorporation and/or pattern or rate ofchange in content and/or pattern of isotope labeling of the DNA derivedfrom keratinocytes or mammary epithelial cells; d) calculating molecularflux rates in the keratinocyte or mammary epithelial cell productionpathway based on the content and/or pattern or rate of change of contentand/or pattern of isotopic labeling in the DNA derived fromkeratinocytes or mammary epithelial cells to monitor or diagnose caloricrestriction/longevity regimens.

Hyperlipoproteinemia or treatment thereof can be monitored or diagnosedby measuring or detecting hepatic bile acid dynamics. In this method,the targeted molecule of interest is one or more hepatic bile acids. Inthis format, the present application is further directed to a method formonitoring or diagnosing hyperlipoproteinemia in a living system, themethod including: a) administering an isotope-labeled substrate to theliving system for a period of time sufficient for the isotope-labeledsubstrate to enter into the hepatic bile acid synthesis pathway andthereby enter into and label at least one hepatic bile acid within thehepatic bile acid synthesis pathway in the living system; b) obtainingone or more samples from the living system, wherein the one or moresamples include at least one isotope-labeled hepatic bile acid; c)measuring the content, rate of incorporation and/or pattern or rate ofchange in content and/or pattern of isotope labeling of the hepatic bileacid; d) calculating molecular flux rates in the hepatic bile acidsynthesis pathway based on the content and/or pattern or rate of changeof content and/or pattern of isotopic labeling in the hepatic bile acidto monitor or diagnose hyperlipoproteinemia or treatment thereof.

Hyperlipoproteinemia or cirrhosis/steatosis risk or treatment thereofcan be monitored or diagnosed by measuring or detecting the dynamics ofconversion of ethanol to acetate and triglyceride. In this method, thetargeted molecule of interest is one or more fatty acids or acetate. Inthis format, the present application is further directed to a method formonitoring or diagnosing hyperlipoproteinemia or cirrhosis/steatosisrisk in a living system, the method including: a) administering anisotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into the pathwayof conversion of ethanol to acetate and triglyceride and thereby enterinto and label at least one fatty acid or acetate within the pathway ofconversion of ethanol to acetate and triglyceride in the living system;b) obtaining one or more samples from the living system, wherein the oneor more samples include at least one isotope-labeled fatty acid oracetate; c) measuring the content, rate of incorporation and/or patternor rate of change in content and/or pattern of isotope labeling of thefatty acid or acetate; d) calculating molecular flux rates in thepathway of conversion of ethanol to acetate and triglyceride based onthe content and/or pattern or rate of change of content and/or patternof isotopic labeling in the fatty acid or acetate to monitor or diagnosehyperlipoproteinemia or cirrhosis/steatosis risk or treatment thereof.

Coronary artery disease risk or treatment of coronary artery disease canbe monitored or diagnosed by measuring or detecting apolipoprotein Bdynamics. In this method, the targeted molecule of interest isapolipoprotein B. In this format, the present application is furtherdirected to a method for monitoring or diagnosing coronary arterydisease risk in a living system, the method including: a) administeringan isotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into theapolipoprotein B metabolic pathway and thereby enter into and label atleast one apolipoprotein B molecule within the apolipoprotein Bmetabolic pathway in the living system; b) obtaining one or more samplesfrom the living system, wherein the one or more samples include at leastone isotope-labeled apolipoprotein B molecule; c) measuring the content,rate of incorporation and/or pattern or rate of change in content and/orpattern of isotope labeling of the apolipoprotein B; d) calculatingmolecular flux rates in the apolipoprotein B metabolic pathway based onthe content and/or pattern or rate of change of content and/or patternof isotopic labeling in the apolipoprotein B to monitor or diagnosecoronary artery disease risk or treatment of coronary artery disease.

Coronary artery disease risk, pancreatitis or hyperlipoproteinemia ortreatment thereof can be monitored or diagnosed by measuring ordetecting very low density lipoprotein (VLDL)-triglyceride dynamics. Inthis method, the targeted molecules of interest are Apolipoprotein B andtriglyceride glycerol. In this format, the present application isfurther directed to a method for monitoring or diagnosing coronaryartery disease risk, pancreatitis or hyperlipoproteinemia in a livingsystem, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the VLDL/triglyceride metabolicpathway and thereby enter into and label at least one Apolipoprotein Bor triglyceride glycerol molecule within the VLDL/triglyceride metabolicpathway in the living system; b) obtaining one or more samples from theliving system, wherein the one or more samples include at least oneisotope-labeled Apolipoprotein B or triglyceride glycerol molecule; c)measuring the content, rate of incorporation and/or pattern or rate ofchange in content and/or pattern of isotope labeling of theApolipoprotein B and triglyceride glycerol; d) calculating molecularflux rates in the VLDL/triglyceride metabolic pathway based on thecontent and/or pattern or rate of change of content and/or pattern ofisotopic labeling in the Apolipoprotein B and triglyceride glycerol tomonitor or diagnose coronary artery disease risk, pancreatitis orhyperlipoproteinemia or treatment thereof.

Statin response or coronary artery disease risk can be monitored ordiagnosed by measuring or detecting cholesterol dynamics. In thismethod, the targeted molecule of interest is cholesterol from serum orblood. In this format, the present application is further directed to amethod for monitoring or diagnosing statin response or coronary arterydisease risk in a living system, the method including: a) administeringan isotope-labeled substrate to the living system in the presence orabsence of a statin for a period of time sufficient for theisotope-labeled substrate to enter into the cholesterol synthesispathway and thereby enter into and label at least one cholesterolmolecule from serum or blood within the cholesterol synthesis pathway inthe living system; b) obtaining one or more samples from the livingsystem, wherein the one or more samples include at least oneisotope-labeled cholesterol molecule from serum or blood; c) measuringthe content, rate of incorporation and/or pattern or rate of change incontent and/or pattern of isotope labeling of the cholesterol from serumor blood; d) calculating molecular flux rates in the cholesterolsynthesis pathway based on the content and/or pattern or rate of changeof content and/or pattern of isotopic labeling in the cholesterol fromserum or blood to monitor or diagnose statin response or coronary arterydisease risk.

Atherosclerosis risk or treatment of atherosclerosis can be monitored ordiagnosed by measuring or detecting vascular smooth muscle celldynamics. In this method, the targeted molecule of interest is DNAderived from vascular smooth muscle cells. In this format, the presentapplication is further directed to a method for monitoring or diagnosingatherosclerosis risk in a living system, the method including: a)administering an isotope-labeled substrate to the living system for aperiod of time sufficient for the isotope-labeled substrate to enterinto the vascular smooth muscle cell production pathway and therebyenter into and label at least one DNA molecule derived from vascularsmooth muscle cells within the vascular smooth muscle cell productionpathway in the living system; b) obtaining one or more samples from theliving system, wherein the one or more samples include at least oneisotope-labeled DNA molecule derived from vascular smooth muscle cells;c) measuring the content, rate of incorporation and/or pattern or rateof change in content and/or pattern of isotope labeling of the DNAderived from vascular smooth muscle cells; d) calculating molecular fluxrates in the vascular smooth muscle cell production pathway based on thecontent and/or pattern or rate of change of content and/or pattern ofisotopic labeling in the DNA derived from vascular smooth muscle cellsto monitor or diagnose atherosclerosis risk or treatment ofatherosclerosis.

Coronary artery disease risk or treatment thereof can be monitored ordiagnosed by measuring or detecting cholesterol transport dynamics(reverse cholesterol transport). In this method, the targeted moleculesof interest are bile acids and cholesterol. In this format, the presentapplication is further directed to a method for monitoring or diagnosingcoronary artery disease risk in a living system, the method including:a) administering an isotope-labeled substrate to the living system for aperiod of time sufficient for the isotope-labeled substrate to enterinto the reverse cholesterol transport pathway and thereby enter intoand label at least one bile acid and cholesterol molecule within thereverse cholesterol transport pathway in the living system; b) obtainingone or more samples from the living system, wherein the one or moresamples include at least one isotope-labeled bile acid and cholesterolmolecule; c) measuring the content, rate of incorporation and/or patternor rate of change in content and/or pattern of isotope labeling of thebile acids and cholesterol; d) calculating molecular flux rates in thereverse cholesterol transport pathway based on the content and/orpattern or rate of change of content and/or pattern of isotopic labelingin the bile acids and cholesterol to monitor or diagnose coronary arterydisease risk.

Cardiomyopathy or treatment thereof can be monitored or diagnosed bymeasuring or detecting cardiac muscle protein dynamics. In this method,the targeted molecule of interest is protein derived from cardiacmuscle. In this format, the present application is further directed to amethod for monitoring or diagnosing cardiomyopathy in a living system,the method including: a) administering an isotope-labeled substrate tothe living system for a period of time sufficient for theisotope-labeled substrate to enter into the cardiac muscle proteinsynthesis pathway and thereby enter into and label at least one proteinderived from cardiac muscle within the cardiac muscle protein synthesispathway in the living system; b) obtaining one or more samples from theliving system, wherein the one or more samples include at least oneisotope-labeled protein derived from cardiac muscle; c) measuring thecontent, rate of incorporation and/or pattern or rate of change incontent and/or pattern of isotope labeling of the protein derived fromcardiac muscle; d) calculating molecular flux rates in the cardiacmuscle protein synthesis pathway based on the content and/or pattern orrate of change of content and/or pattern of isotopic labeling in theprotein derived from cardiac muscle to monitor or diagnosecardiomyopathy or treatment thereof.

Cardiac fitness or congestive heart failure or treatment thereof can bemonitored or diagnosed by measuring or detecting cardiac collagendynamics. In this method, the targeted molecule of interest is collagenderived from cardiac tissue. In this format, the present application isfurther directed to a method for monitoring or diagnosing cardiacfitness or congestive heart failure in a living system, the methodincluding: a) administering an isotope-labeled substrate to the livingsystem for a period of time sufficient for the isotope-labeled substrateto enter into the cardiac collagen synthesis pathway and thereby enterinto and label at least one collagen molecule derived from cardiactissue within the cardiac collagen synthesis pathway in the livingsystem; b) obtaining one or more samples from the living system, whereinthe one or more samples include at least one isotope-labeled collagenmolecule derived from cardiac tissue; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the collagen derived from cardiac tissue; d)calculating molecular flux rates in the cardiac collagen synthesispathway based on the content and/or pattern or rate of change of contentand/or pattern of isotopic labeling in the collagen derived from cardiactissue to monitor or diagnose cardiac fitness or congestive heartfailure or treatment thereof.

Vasculitis or treatment thereof can be monitored or diagnosed bymeasuring or detecting vascular smooth muscle cell or endothelial celldynamics. In this method, the targeted molecule of interest is DNAderived from vascular smooth muscle cells or endothelial cells. In thisformat, the present application is further directed to a method formonitoring or diagnosing vasculitis in a living system, the methodincluding: a) administering an isotope-labeled substrate to the livingsystem for a period of time sufficient for the isotope-labeled substrateto enter into the vascular smooth muscle cell or endothelial cellproduction pathway and thereby enter into and label at least one DNAmolecule derived from vascular smooth muscle cells or endothelial cellswithin the vascular smooth muscle cell or endothelial cell productionpathway in the living system; b) obtaining one or more samples from theliving system, wherein the one or more samples include at least oneisotope-labeled DNA molecule derived from vascular smooth muscle cellsor endothelial cells; c) measuring the content, rate of incorporationand/or pattern or rate of change in content and/or pattern of isotopelabeling of the DNA derived from vascular smooth muscle cells orendothelial cells; d) calculating molecular flux rates in the vascularsmooth muscle cell or endothelial cell production pathway based on thecontent and/or pattern or rate of change of content and/or pattern ofisotopic labeling in the DNA derived from vascular smooth muscle cellsor endothelial cells to monitor or diagnose vasculitis or treatmentthereto.

Psoriasis, skin hyperproliferation or ectopy or response of treatmentthereto with one or more compounds can be monitored or diagnosed bymeasuring or detecting keratinocyte dynamics. In this method, thetargeted molecule of interest is DNA derived from keratinocytes. In thisformat, the present application is further directed to a method formonitoring or diagnosing psoriasis, skin hyperproliferation or ectopy ina living system, the method including: a) administering anisotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into thekeratinocyte production pathway and thereby enter into and label atleast one DNA molecule derived from keratinocytes within thekeratinocyte production pathway in the living system; b) obtaining oneor more samples from the living system, wherein the one or more samplesinclude at least one isotope-labeled DNA molecule derived fromkeratinocytes; c) measuring the content, rate of incorporation and/orpattern or rate of change in content and/or pattern of isotope labelingof the DNA derived from keratinocytes; d) calculating molecular fluxrates in the keratinocyte production pathway based on the content and/orpattern or rate of change of content and/or pattern of isotopic labelingin the DNA derived from keratinocytes to monitor or diagnose psoriasis,skin hyperproliferation or ectopy or response of treatment thereto withone or more compounds.

Psoriasis or skin barrier can be monitored or diagnosed by measuring ordetecting skin keratin dynamics. In this method, the targeted moleculeof interest is skin keratin. In this format, the present application isfurther directed to a method for monitoring or diagnosing psoriasis orskin barrier in a living system, the method including: a) administeringan isotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into the skinkeratin synthesis pathway and thereby enter into and label at least oneskin keratin molecule within the skin keratin synthesis pathway in theliving system; b) obtaining one or more samples from the living system,wherein the one or more samples include at least one isotope-labeledskin keratin molecule; c) measuring the content, rate of incorporationand/or pattern or rate of change in content and/or pattern of isotopelabeling of the skin keratin; d) calculating molecular flux rates in theskin keratin synthesis pathway based on the content and/or pattern orrate of change of content and/or pattern of isotopic labeling in theskin keratin to monitor or diagnose psoriasis or skin barrier.

Skin wrinkles, dermatomyolitis or scleroderma or response to treatmentof same with one or more compounds can be monitored or diagnosed bymeasuring or detecting skin collagen dynamics and elastin dynamics. Inthis method, the targeted molecule of interest is collagen from skin(epidermis or dermis). In this format, the present application isfurther directed to a method for monitoring or diagnosing skin wrinkles,dermatomyolitis or scleroderma in a living system, the method including:a) administering an isotope-labeled substrate to the living system for aperiod of time sufficient for the isotope-labeled substrate to enterinto the skin collagen and elastin synthesis pathways and thereby enterinto and label at least one collagen molecule from skin within the skincollagen and elastin synthesis pathways in the living system; b)obtaining one or more samples from the living system, wherein the one ormore samples include at least one isotope-labeled collagen molecule fromskin; c) measuring the content, rate of incorporation and/or pattern orrate of change in content and/or pattern of isotope labeling of thecollagen from skin; d) calculating molecular flux rates in the skincollagen and elastin synthesis pathways based on the content and/orpattern or rate of change of content and/or pattern of isotopic labelingin the collagen from skin to monitor or diagnose skin wrinkles,dermatomyolitis or scleroderma or response to treatment of same with oneor more compounds.

Wound healing, adjunctive compound or therapeutic response to treatmentthereto within one or more compounds can be monitored or diagnosed bymeasuring or detecting wound collagen dynamics. In this method, thetargeted molecule of interest is collagen from skin and other woundedtissues. In this format, the present application is further directed toa method for monitoring or diagnosing wound healing, adjunctive compoundor therapeutic response in a living system, the method including: a)administering an isotope-labeled substrate to the living system for aperiod of time sufficient for the isotope-labeled substrate to enterinto the wound collagen production pathway and thereby enter into andlabel at least one collagen molecule from skin and other wounded tissueswithin the wound collagen production pathway in the living system; b)obtaining one or more samples from the living system, wherein the one ormore samples include at least one isotope-labeled collagen molecule fromskin and other wounded tissues; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the collagen from skin and other wounded tissues;d) calculating molecular flux rates in the wound collagen productionpathway based on the content and/or pattern or rate of change of contentand/or pattern of isotopic labeling in the collagen from skin and otherwounded tissues to monitor or diagnose wound healing, adjunctivecompound or therapeutic response to treatment thereto.

Osteoarthritis, rheumatoid arthritis, joint protection/destruction ordiet or response to treatment of same with one or more compounds can bemonitored or diagnosed by measuring or detecting synovial spacehyaluronic acid or chondroitin sulfate dynamics. In this method, thetargeted molecules of interest are hyaluronic acid from synovial fluidor cartilage and chondroitin sulfate from synovial fluid or cartilage.In this format, the present application is further directed to a methodfor monitoring or diagnosing osteoarthritis, rheumatoid arthritis, jointprotection/destruction or diet in a living system, the method including:a) administering an isotope-labeled substrate to the living system for aperiod of time sufficient for the isotope-labeled substrate to enterinto the synovial space hyaluronic acid or chondroitin sulfate pathwaysand thereby enter into and label at least one hyaluronic acid fromsynovial fluid or cartilage and one chondroitin sulfate molecule fromsynovial fluid or cartilage within the synovial space hyaluronic acid orchondroitin sulfate pathways in the living system; b) obtaining one ormore samples from the living system, wherein the one or more samplesinclude at least one isotope-labeled hyaluronic acid from synovial fluidor cartilage and one chondroitin sulfate molecule from synovial fluid orcartilage; c) measuring the content, rate of incorporation and/orpattern or rate of change in content and/or pattern of isotope labelingof the hyaluronic acid from synovial fluid or cartilage and chondroitinsulfate from synovial fluid or cartilage; d) calculating molecular fluxrates in the synovial space hyaluronic acid or chondroitin sulfatepathways based on the content and/or pattern or rate of change ofcontent and/or pattern of isotopic labeling in the hyaluronic acid fromsynovial fluid or cartilage and chondroitin sulfate from synovial fluidor cartilage to monitor or diagnose osteoarthritis, rheumatoidarthritis, joint protection/destruction or diet.

Osteoporosis, pagets or healing of bone fractures or response totreatment of same with one or more compounds can be monitored ordiagnosed by measuring or detecting bone collagen dynamics. In thismethod, the targeted molecule of interest is collagen from bone. In thisformat, the present application is further directed to a method formonitoring or diagnosing osteoporosis, pagets or healing of bonefractures in a living system, the method including: a) administering anisotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into the bonecollagen synthesis pathway and thereby enter into and label at least onecollagen molecule from bone within the bone collagen synthesis pathwayin the living system; b) obtaining one or more samples from the livingsystem, wherein the one or more samples include at least oneisotope-labeled collagen molecule from bone; c) measuring the content,rate of incorporation and/or pattern or rate of change in content and/orpattern of isotope labeling of the collagen from bone; d) calculatingmolecular flux rates in the bone collagen synthesis pathway based on thecontent and/or pattern or rate of change of content and/or pattern ofisotopic labeling in the collagen from bone to monitor or diagnoseosteoporosis, pagets or healing of bone fractures or response totreatment of same with one or more compounds.

Osteoarthritis, rheumatoid arthritis, joint protection or response totreatment of same with one or more compounds can be monitored ordiagnosed by measuring or detecting joint collagen dynamics. In thismethod, the targeted molecule of interest is collagen from synovialfluid or cartilage. In this format, the present application is furtherdirected to a method for monitoring or diagnosing osteoarthritis,rheumatoid arthritis, joint protection or response to treatment in aliving system, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the joint collagen synthesispathway and thereby enter into and label at least one collagen moleculefrom synovial fluid or cartilage within the joint collagen synthesispathway in the living system; b) obtaining one or more samples from theliving system, wherein the one or more samples include at least oneisotope-labeled collagen molecule from synovial fluid or cartilage; c)measuring the content, rate of incorporation and/or pattern or rate ofchange in content and/or pattern of isotope labeling of the collagenfrom synovial fluid or cartilage; d) calculating molecular flux rates inthe joint collagen synthesis pathway based on the content and/or patternor rate of change of content and/or pattern of isotopic labeling in thecollagen from synovial fluid or cartilage to monitor or diagnoseosteoarthritis, rheumatoid arthritis, joint protection or response totreatment of same with one or more compounds.

Rheumatoid arthritis or joint destruction or response to treatment ofsame with one or more compounds can be monitored or diagnosed bymeasuring or detecting synovial leukocyte/T-cell dynamics. In thismethod, the targeted molecule of interest is DNA from leukocytes orT-cells in synovial fluid or associated with joints. In this format, thepresent application is further directed to a method for monitoring ordiagnosing rheumatoid arthritis or joint destruction in a living system,the method including: a) administering an isotope-labeled substrate tothe living system for a period of time sufficient for theisotope-labeled substrate to enter into the synovial leukocyte/T-cellproduction pathways and thereby enter into and label at least one DNAmolecule from leukocytes or T-cells in synovial fluid or associated withjoints within the synovial leukocyte/T-cell production pathways in theliving system; b) obtaining one or more samples from the living system,wherein the one or more samples include at least one isotope-labeled DNAmolecule from leukocytes or T-cells in synovial fluid or associated withjoints; c) measuring the content, rate of incorporation and/or patternor rate of change in content and/or pattern of isotope labeling of theDNA from leukocytes or T-cells in synovial fluid or associated withjoints; d) calculating molecular flux rates in the synovialleukocyte/T-cell production pathways based on the content and/or patternor rate of change of content and/or pattern of isotopic labeling in theDNA from leukocytes or T-cells in synovial fluid or associated withjoints to monitor or diagnose rheumatoid arthritis or joint destructionor response to treatment thereof.

Risk for cancer or therapeutic response to a treatment thereof can bemonitored or diagnosed by measuring or detecting mammary epithelial celldynamics. In this method, the targeted molecule of interest is DNA frommammary epithelial cells. In this format, the present application isfurther directed to a method for monitoring or diagnosing risk forcancer or compound or therapeutic response in a living system, themethod including: a) administering an isotope-labeled substrate to theliving system for a period of time sufficient for the isotope-labeledsubstrate to enter into the mammary epithelial cell production pathwayand thereby enter into and label at least one DNA molecule from mammaryepithelial cells within the mammary epithelial cell production pathwayin the living system; b) obtaining one or more samples from the livingsystem, wherein the one or more samples include at least oneisotope-labeled DNA molecule from mammary epithelial cells; c) measuringthe content, rate of incorporation and/or pattern or rate of change incontent and/or pattern of isotope labeling of the DNA from mammaryepithelial cells; d) calculating molecular flux rates in the mammaryepithelial cell production pathway based on the content and/or patternor rate of change of content and/or pattern of isotopic labeling in theDNA from mammary epithelial cells to monitor or diagnose risk for canceror therapeutic treatment thereof.

Risk for cancer or therapeutic treatment thereof can be monitored ordiagnosed by measuring or detecting colon epithelial cell dynamics. Inthis method, the targeted molecule of interest is DNA from colonepithelial cells. In this format, the present application is furtherdirected to a method for monitoring or diagnosing risk for cancer orcompound or therapeutic response in a living system, the methodincluding: a) administering an isotope-labeled substrate to the livingsystem for a period of time sufficient for the isotope-labeled substrateto enter into the colon epithelial cell production pathway and therebyenter into and label at least one DNA molecule from colon epithelialcells within the colon epithelial cell production pathway in the livingsystem; b) obtaining one or more samples from the living system, whereinthe one or more samples include at least one isotope-labeled DNAmolecule from colon epithelial cells; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the DNA from colon epithelial cells; d)calculating molecular flux rates in the colon epithelial cell productionpathway based on the content and/or pattern or rate of change of contentand/or pattern of isotopic labeling in the DNA from colon epithelialcells to monitor or diagnose risk for cancer or compound or therapeuticresponse.

Risk for cancer or therapeutic treatment thereof can be monitored ordiagnosed by measuring or detecting bronchial cell or tissue dynamics.In this method, the targeted molecule of interest is DNA from bronchialtissue. In this format, the present application is further directed to amethod for monitoring or diagnosing risk for cancer or compound ortherapeutic response in a living system, the method including: a)administering an isotope-labeled substrate to the living system for aperiod of time sufficient for the isotope-labeled substrate to enterinto the bronchial cell or tissue production pathway and thereby enterinto and label at least one DNA molecule from bronchial tissue withinthe bronchial cell or tissue production pathway in the living system; b)obtaining one or more samples from the living system, wherein the one ormore samples include at least one isotope-labeled DNA molecule frombronchial tissue; c) measuring the content, rate of incorporation and/orpattern or rate of change in content and/or pattern of isotope labelingof the DNA from bronchial tissue; d) calculating molecular flux rates inthe bronchial cell or tissue production pathway based on the contentand/or pattern or rate of change of content and/or pattern of isotopiclabeling in the DNA from bronchial tissue to monitor or diagnose riskfor cancer or therapeutic treatment thereof.

Risk for cancer, benign prostatic hyperplasia or therapeutic thereof canbe monitored or diagnosed by measuring or detecting prostate epithelialcell dynamics. In this method, the targeted molecule of interest is DNAfrom prostate epithelial cells. In this format, the present applicationis further directed to a method for monitoring or diagnosing risk forcancer, benign prostatic hyperplasia or compound or therapeutic responsein a living system, the method including: a) administering anisotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into the prostateepithelial cell production pathway and thereby enter into and label atleast one DNA molecule from prostate epithelial cells within theprostate epithelial cell production pathway in the living system; b)obtaining one or more samples from the living system, wherein the one ormore samples include at least one isotope-labeled DNA molecule fromprostate epithelial cells; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the DNA from prostate epithelial cells; d)calculating molecular flux rates in the prostate epithelial cellproduction pathway based on the content and/or pattern or rate of changeof content and/or pattern of isotopic labeling in the DNA from prostateepithelial cells to monitor or diagnose risk for cancer, benignprostatic hyperplasia or therapeutic treatment thereof.

Risk for cancer or therapeutic treatment thereof can be monitored ordiagnosed by measuring or detecting the dynamics of tumors of pancreas,bladder, gastric, brain, ovary, or cervix. In this method, the targetedmolecule of interest is DNA from cells from which tumors may derive(e.g., epithelial cells) or pre-cancerous cells, or cells whoseproliferative behavior is associated with increased risk of cancer. Inthis format, the present application is further directed to a method formonitoring or diagnosing risk for cancer or compound or therapeuticresponse in a living system, the method including: a) administering anisotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into thetumorigenesis pathway of pancreas, bladder, gastric, brain, ovary, orcervix cancer and thereby enter into and label at least one DNA moleculefrom cells from which tumors may derive from the tumorigenesis ofpancreas, bladder, gastric, brain, ovary, or cervix in the livingsystem; b) obtaining one or more samples from the living system, whereinthe one or more samples include at least one isotope-labeled DNAmolecule from cells from which tumors may derive; c) measuring thecontent, rate of incorporation and/or pattern or rate of change incontent and/or pattern of isotope labeling of the DNA from cells fromwhich tumors may derive; d) calculating molecular flux rates in thetumorigenesis of pancreas, bladder, gastric, brain, ovary, or cervixcancer based on the content and/or pattern or rate of change of contentand/or pattern of isotopic labeling in the DNA from cells from whichtumors may derive to monitor or diagnose risk for cancer or therapeutictreatment thereof.

Tumor growth, grade, prognosis, aggressiveness, or therapeutic treatmentthereof can be monitored or diagnosed by measuring or detecting thedynamics of solid tumors (including breast, colon, lung, and lymphoma).In this method, the targeted molecule of interest is DNA derived fromsolid tumor cells. In this format, the present application is furtherdirected to a method for monitoring or diagnosing tumor growth, grade,prognosis, aggressiveness, or therapeutic response in a living system,the method including: a) administering an isotope-labeled substrate tothe living system for a period of time sufficient for theisotope-labeled substrate to enter into the solid tumor formationpathway and thereby enter into and label at least one DNA moleculederived from solid tumor cells within the solid tumor formation pathwayin the living system; b) obtaining one or more samples from the livingsystem, wherein the one or more samples include at least oneisotope-labeled DNA molecule derived from solid tumor cells; c)measuring the content, rate of incorporation and/or pattern or rate ofchange in content and/or pattern of isotope labeling of the DNA derivedfrom solid tumor cells; d) calculating molecular flux rates in the solidtumor formation pathway based on the content and/or pattern or rate ofchange of content and/or pattern of isotopic labeling in the DNA derivedfrom solid tumor cells to monitor or diagnose tumor growth, grade,prognosis, aggressiveness, or therapeutic treatment thereof.

Cancer growth, prognosis, or therapeutic treatment thereof can bemonitored or diagnosed by measuring or detecting the dynamics of liquidtumors. In this method, the targeted molecule of interest is DNA derivedfrom liquid tumor cells. In this format, the present application isfurther directed to a method for monitoring or diagnosing cancer growth,prognosis, or compound or therapeutic response in a living system, themethod including: a) administering an isotope-labeled substrate to theliving system for a period of time sufficient for the isotope-labeledsubstrate to enter into the liquid tumor formation pathway and therebyenter into and label at least one DNA molecule derived from liquid tumorcells within the liquid tumor formation pathway in the living system; b)obtaining one or more samples from the living system, wherein the one ormore samples include at least one isotope-labeled DNA molecule derivedfrom liquid tumor cells; c) measuring the content, rate of incorporationand/or pattern or rate of change in content and/or pattern of isotopelabeling of the DNA derived from liquid tumor cells; d) calculatingmolecular flux rates in the liquid tumor formation pathway based on thecontent and/or pattern or rate of change of content and/or pattern ofisotopic labeling in the DNA derived from liquid tumor cells to monitoror diagnose cancer growth, prognosis, or therapeutic treatment thereof.

Multiple myeloma activity, prognosis, growth, mass or therapeutictreatment thereof can be monitored or diagnosed by measuring ordetecting immunoglobulin, albumin, myeloma-protein dynamics or myelomacell dynamics. In this method, the targeted molecule of interest ismyeloma protein, immunoglobulin or albumin derived from serum or bonemarrow, or DNA from myeloma cells. In this format, the presentapplication is further directed to a method for monitoring or diagnosingmultiple myeloma activity, prognosis, growth, mass or therapeuticresponse to treatment thereof in a living system, the method including:a) administering an isotope-labeled substrate to the living system for aperiod of time sufficient for the isotope-labeled substrate to enterinto the immunoglobulin, albumin, myeloma-protein or myeloma cellproduction pathways and thereby enter into and label at least onemyeloma protein, immunoglobulin, or albumin derived from serum or bonemarrow, or one DNA molecule from myeloma cells within theimmunoglobulin, albumin, myeloma-protein or myeloma cell productionpathways in the living system; b) obtaining one or more samples from theliving system, wherein the one or more samples include at least oneisotope-labeled myeloma protein, immunoglobulin, or albumin derived fromserum or bone marrow, or one DNA molecule from myeloma cells; c)measuring the content, rate of incorporation and/or pattern or rate ofchange in content and/or pattern of isotope labeling of the myelomaprotein, immunoglobulin, or albumin derived from serum or bone marrow,or DNA from myeloma cells; d) calculating molecular flux rates in theimmunoglobulin, albumin, myeloma-protein or myeloma cell productionpathways based on the content and/or pattern or rate of change ofcontent and/or pattern of isotopic labeling in the myeloma protein,immunoglobulin, or albumin derived from serum or bone marrow, or DNAfrom myeloma cells to monitor or diagnose multiple myeloma activity,prognosis, growth, mass or therapeutic treatment thereof.

Angiogenesis or therapeutic treatment thereof can be monitored ordiagnosed by measuring or detecting tumor endothelial cell dynamics. Inthis method, the targeted molecule of interest is DNA from tumorendothelial cells. In this format, the present application is furtherdirected to a method for monitoring or diagnosing angiogenesis orcompound or therapeutic response in a living system, the methodincluding: a) administering an isotope-labeled substrate to the livingsystem for a period of time sufficient for the isotope-labeled substrateto enter into the tumor endothelial cell production pathway and therebyenter into and label at least one DNA molecule from tumor endothelialcells within the tumor endothelial cell production pathway in the livingsystem; b) obtaining one or more samples from the living system, whereinthe one or more samples include at least one isotope-labeled DNAmolecule from tumor endothelial cells; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the DNA from tumor endothelial cells; d)calculating molecular flux rates in the tumor endothelial cellproduction pathway based on the content and/or pattern or rate of changeof content and/or pattern of isotopic labeling in the DNA from tumorendothelial cells to monitor or diagnose angiogenesis or therapeutictreatment thereof.

Angiogenesis or therapeutic treatment thereof can be monitored ordiagnosed by measuring or detecting the dynamics of ribonucleotidereductase substrates and metabolites (flux vs. salvage). In this method,the targeted molecule of interest is deoxyadenosine and deoxythymidine.In this format, the present application is further directed to a methodfor monitoring or diagnosing compound or therapeutic response in aliving system, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the metabolism of ribonucleotidereductase substrates and thereby enter into and label at least onemolecule of deoxyadenosine and deoxythymidine within the metabolism ofribonucleotide reductase substrates in the living system; b) obtainingone or more samples from the living system, wherein the one or moresamples include at least one isotope-labeled molecule of deoxyadenosineand deoxythymidine; c) measuring the content, rate of incorporationand/or pattern or rate of change in content and/or pattern of isotopelabeling of the deoxyadenosine and deoxythymidine; d) calculatingmolecular flux rates in the metabolism of ribonucleotide reductasesubstrates based on the content and/or pattern or rate of change ofcontent and/or pattern of isotopic labeling in the deoxyadenosine anddeoxythymidine to monitor or diagnose angiogenesis or therapeuticresponse to treatment thereof.

Cancer risk or therapeutic response to treatment thereto can bemonitored or diagnosed by measuring or detecting epithelial stem celldynamics. In this method, the targeted molecule of interest is DNA fromepithelial stem cells. In this format, the present application isfurther directed to a method for monitoring or diagnosing cancer risk orcompound or therapeutic response in a living system, the methodincluding: a) administering an isotope-labeled substrate to the livingsystem for a period of time sufficient for the isotope-labeled substrateto enter into the epithelial stem cell production pathway and therebyenter into and label at least one DNA molecule from epithelial stemcells within the epithelial stem cell production pathway in the livingsystem; b) obtaining one or more samples from the living system, whereinthe one or more samples include at least one isotope-labeled DNAmolecule from epithelial stem cells; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the DNA from epithelial stem cells; d)calculating molecular flux rates in the epithelial stem cell productionpathway based on the content and/or pattern or rate of change of contentand/or pattern of isotopic labeling in the DNA from epithelial stemcells to monitor or diagnose cancer risk or therapeutic response totreatment thereto.

Tumor grade, prognosis, treatment target, or therapeutic response totreatment thereto can be monitored or diagnosed by measuring ordetecting tumor cell RNA dynamics. In this method, the targeted moleculeof interest is RNA from tumor cells, either total ortranscript-specific. In this format, the present application is furtherdirected to a method for monitoring or diagnosing tumor grade,prognosis, treatment target, or compound or therapeutic response in aliving system, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into tumor cell transcription andthereby enter into and label at least one RNA molecule from tumor cellswithin tumor cell transcription in the living system; b) obtaining oneor more samples from the living system, wherein the one or more samplesinclude at least one isotope-labeled RNA molecule from tumor cells; c)measuring the content, rate of incorporation and/or pattern or rate ofchange in content and/or pattern of isotope labeling of the RNA fromtumor cells; d) calculating molecular flux rates in tumor celltranscription based on the content and/or pattern or rate of change ofcontent and/or pattern of isotopic labeling in the RNA from tumor cellsto monitor or diagnose tumor grade, prognosis, treatment target, ortherapeutic response to treatment thereto.

Proliferation and growth of transplant can be monitored or diagnosed bymeasuring or detecting T-cell or other blood cell dynamics (post bonemarrow transplant). In this method, the targeted molecule of interest isDNA from transplanted cells, or from cells maturing from transplantedcells. In this format, the present application is further directed to amethod for monitoring or diagnosing proliferation and growth oftransplant in a living system, the method including: a) administering anisotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into the T-cell orother blood cell production pathway and thereby enter into and label atleast one DNA molecule from transplanted cells, or from cells maturingfrom transplanted cells within the T-cell or other blood cell productionpathway in the living system; b) obtaining one or more samples from theliving system, wherein the one or more samples include at least oneisotope-labeled DNA molecule from transplanted cells, or from cellsmaturing from transplanted cells; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the DNA from transplanted cells, or from cellsmaturing from transplanted cells; d) calculating molecular flux rates inthe T-cell or other blood cell production pathway based on the contentand/or pattern or rate of change of content and/or pattern of isotopiclabeling in the DNA from transplanted cells, or from cells maturing fromtransplanted cells to monitor or diagnose proliferation and growth oftransplant.

Adequacy of surgery can be monitored or diagnosed by measuring ordetecting cell dynamics at the surgical margin of a tumor. In thismethod, the targeted molecule of interest is DNA from the surgicalmargin of the tumor. In this format, the present application is furtherdirected to a method for monitoring or diagnosing adequacy of surgery ina living system, the method including: a) administering anisotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into the pathwaywhereby cells are formed at the surgical margin of a tumor and therebyenter into and label at least one DNA molecule from the surgical marginof the tumor within the pathway whereby cells are formed at the surgicalmargin of a tumor in the living system; b) obtaining one or more samplesfrom the living system, wherein the one or more samples include at leastone isotope-labeled DNA molecule from the surgical margin of the tumor;c) measuring the content, rate of incorporation and/or pattern or rateof change in content and/or pattern of isotope labeling of the DNA fromthe surgical margin of the tumor; d) calculating molecular flux rates inthe pathway whereby cells are formed at the surgical margin of a tumorbased on the content and/or pattern or rate of change of content and/orpattern of isotopic labeling in the DNA from the surgical margin of thetumor to monitor or diagnose adequacy of surgery.

Grade, aggressiveness, or graft-versus-host-disease treatment responsecan be monitored or diagnosed by measuring or detecting grafted tissuedynamics. In this method, the targeted molecule of interest is DNA fromthe grafted tissue. In this format, the present application is furtherdirected to a method for monitoring or diagnosing grade, aggressiveness,or graft-versus-host-disease treatment response in a living system, themethod including: a) administering an isotope-labeled substrate to theliving system for a period of time sufficient for the isotope-labeledsubstrate to enter into the pathway of incorporation of grafted tissueand thereby enter into and label at least one DNA molecule from thegrafted tissue within the pathway of incorporation of grafted tissue inthe living system; b) obtaining one or more samples from the livingsystem, wherein the one or more samples include at least oneisotope-labeled DNA molecule from the grafted tissue; c) measuring thecontent, rate of incorporation and/or pattern or rate of change incontent and/or pattern of isotope labeling of the DNA from the graftedtissue; d) calculating molecular flux rates in the pathway ofincorporation of grafted tissue based on the content and/or pattern orrate of change of content and/or pattern of isotopic labeling in the DNAfrom the grafted tissue to monitor or diagnose grade, aggressiveness, orgraft-versus-host-disease treatment response.

Diagnosis of cancer, monitoring of cancer progression and treatment ofcancer by gene silencing can be monitored or diagnosed by measuring ordetecting the dynamics of methylcytosine (methyl deoxycytosinemethylation/hypo methylation). In this method, the targeted molecule ofinterest is methyl deoxycytosine from DNA from cells of interest. Inthis format, the present application is further directed to a method formonitoring or diagnosing gene silencing, prognosis; or compound ortherapeutic response in a living system, the method including: a)administering an isotope-labeled substrate to the living system for aperiod of time sufficient for the isotope-labeled substrate to enterinto the methyl deoxycytosine methylation pathway and thereby enter intoand label at least one methyl deoxycytosine molecule from DNA from cellsof interest within the methyl deoxycytosine methylation pathway in theliving system; b) obtaining one or more samples from the living system,wherein the one or more samples include at least one isotope-labeledmethyl deoxycytosine molecule from DNA from cells of interest; c)measuring the content, rate of incorporation and/or pattern or rate ofchange in content and/or pattern of isotope labeling of the methyldeoxycytosine from DNA from cells of interest; d) calculating molecularflux rates in the methyl deoxycytosine methylation pathway based on thecontent and/or pattern or rate of change of content and/or pattern ofisotopic labeling in the methyl deoxycytosine from DNA from cells ofinterest to monitor or diagnose cancer, or cancer progression or cancertreatments by gene silencing.

Alzheimer's disease risk or response to treatment thereto can bemonitored or diagnosed by measuring or detecting brain amyloid-β oramyloid precursor protein dynamics. In this method, the targetedmolecule of interest is amyloid beta peptide or amyloid precursorprotein or subfragments of either. In this format, the presentapplication is further directed to a method for monitoring or diagnosingAlzheimer's disease risk or response to treatment in a living system,the method including: a) administering an isotope-labeled substrate tothe living system for a period of time sufficient for theisotope-labeled substrate to enter into the brain amyloid-β or amyloidprecursor protein synthesis pathway and thereby enter into and label atleast one amyloid beta peptide or amyloid precursor protein orsubfragments of either within the brain amyloid-β or amyloid precursorprotein synthesis pathway in the living system; b) obtaining one or moresamples from the living system, wherein the one or more samples includeat least one isotope-labeled amyloid beta peptide or amyloid precursorprotein or subfragments of either; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the amyloid beta peptide or amyloid precursorprotein or subfragments of either; d) calculating molecular flux ratesin the brain amyloid-β or amyloid precursor protein synthesis pathwaybased on the content and/or pattern or rate of change of content and/orpattern of isotopic labeling in the amyloid beta peptide or amyloidprecursor protein or subfragments of either to monitor or diagnoseAlzheimer's disease risk or response to treatment thereto.

Multiple sclerosis (MS) activity, MS response to treatment, spinal cordand brain injury recovery or therapeutic response to treatment theretocan be monitored or diagnosed by measuring or detecting brain orperipheral nervous system myelin dynamics. In this method, the targetedmolecule of interest is galactocerebroside from brain, peripheralnervous system, or blood. In this format, the present application isfurther directed to a method for monitoring or diagnosing multiplesclerosis (MS) activity, MS response to treatment, spinal cord and braininjury recovery and/or compound or therapeutic response in a livingsystem, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the brain or peripheral nervoussystem myelin production pathway and thereby enter into and label atleast one galactocerebroside molecule within the brain or peripheralnervous system myelin production pathway in the living system; b)obtaining one or more samples from the living system, wherein the one ormore samples include at least one isotope-labeled galactocerebrosidemolecule; c) measuring the content, rate of incorporation and/or patternor rate of change in content and/or pattern of isotope labeling of thegalactocerebroside; d) calculating molecular flux rates in the brain orperipheral nervous system myelin production pathway based on the contentand/or pattern or rate of change of content and/or pattern of isotopiclabeling in the galactocerebroside to monitor or diagnose multiplesclerosis (MS) activity, MS response to treatment, spinal cord and braininjury recovery or therapeutic response to treatment thereto ortherapeutic response to treatment thereto.

Neurogenesis, x-ray therapy toxicity, development, stress or depressionor therapeutic response to treatment thereto can be monitored ordiagnosed by measuring or detecting neuron dynamics. In this method, thetargeted molecule of interest is DNA from neurons. In this format, thepresent application is further directed to a method for monitoring ordiagnosing neurogenesis, x-ray therapy toxicity, development, stress ordepression in a living system, the method including: a) administering anisotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into theneurogenesis pathway and thereby enter into and label at least one DNAmolecule from neurons within the neurogenesis pathway in the livingsystem; b) obtaining one or more samples from the living system, whereinthe one or more samples include at least one isotope-labeled DNAmolecule from neurons; c) measuring the content, rate of incorporationand/or pattern or rate of change in content and/or pattern of isotopelabeling of the DNA from neurons; d) calculating molecular flux rates inthe neurogenesis pathway based on the content and/or pattern or rate ofchange of content and/or pattern of isotopic labeling in the DNA fromneurons to monitor or diagnose neurogenesis, x-ray therapy toxicity,development, stress or depression or therapeutic response to treatmentthereto.

Psychiatric disorders or treatment thereof can be monitored or diagnosedby measuring or detecting neurotransmitter dynamics. In this method, thetargeted molecules of interest are neurotransmitters from brain, orcirculating or degraded neurotransmitters found in other tissues. Inthis format, the present application is further directed to a method formonitoring or diagnosing psychiatric disorders in a living system, themethod including: a) administering an isotope-labeled substrate to theliving system for a period of time sufficient for the isotope-labeledsubstrate to enter into the neurotransmitter synthesis pathway andthereby enter into and label at least one neurotransmitter within theneurotransmitter synthesis pathway in the living system; b) obtainingone or more samples from the living system, wherein the one or moresamples include at least one isotope-labeled neurotransmitter; c)measuring the content, rate of incorporation and/or pattern or rate ofchange in content and/or pattern of isotope labeling of theneurotransmitter; d) calculating molecular flux rates in theneurotransmitter synthesis pathway based on the content and/or patternor rate of change of content and/or pattern of isotopic labeling in theneurotransmitter to monitor or diagnose psychiatric disorders ortreatment thereof.

Neurogenesis, depression or therapeutic response to treatment theretocan be monitored or diagnosed by measuring or detecting neuroprogenitorcell dynamics. In this method, the targeted molecule of interest is DNAfrom neuroprogenitor cells. In this format, the present application isfurther directed to a method for monitoring or diagnosing neurogenesis,depression or compound or therapeutic response in a living system, themethod including: a) administering an isotope-labeled substrate to theliving system for a period of time sufficient for the isotope-labeledsubstrate to enter into the neuroprogenitor cell production pathway andthereby enter into and label at least one DNA molecule fromneuroprogenitor cells within the neuroprogenitor cell production pathwayin the living system; b) obtaining one or more samples from the livingsystem, wherein the one or more samples include at least oneisotope-labeled DNA molecule from neuroprogenitor cells; c) measuringthe content, rate of incorporation and/or pattern or rate of change incontent and/or pattern of isotope labeling of the DNA fromneuroprogenitor cells; d) calculating molecular flux rates in theneuroprogenitor cell production pathway based on the content and/orpattern or rate of change of content and/or pattern of isotopic labelingin the DNA from neuroprogenitor cells to monitor or diagnoseneurogenesis, depression or therapeutic response to treatment thereto.

Neuroinflammation, multiple sclerosis, Alzheimer's disease, stroke,autism, depression, chronic pain, amyotrophic lateral sclerosis,cerebral amyloid angiopathy, excitotoxic injury or therapeutic responseto treatment thereto can be monitored or diagnosed by measuring ordetecting microglial cell dynamics. In this method, the targetedmolecule of interest is DNA from microglia. In this format, the presentapplication is further directed to a method for monitoring or diagnosingneuroinflammation, multiple sclerosis, Alzheimer's disease, stroke,autism, depression, chronic pain, amyotrophic lateral sclerosis,cerebral amyloid angiopathy, excitotoxic injury or therapeutic responsein a living system, the method including: a) administering anisotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into themicroglial cell production pathway and thereby enter into and label atleast one DNA molecule from microglia within the microglial cellproduction pathway in the living system; b) obtaining one or moresamples from the living system, wherein the one or more samples includeat least one isotope-labeled DNA molecule from microglia; c) measuringthe content, rate of incorporation and/or pattern or rate of change incontent and/or pattern of isotope labeling of the DNA from microglia; d)calculating molecular flux rates in the microglial cell productionpathway based on the content and/or pattern or rate of change of contentand/or pattern of isotopic labeling in the DNA from microglia to monitoror diagnose neuroinflammation, multiple sclerosis, Alzheimer's disease,stroke, autism, depression, chronic pain, amyotrophic lateral sclerosis,cerebral amyloid angiopathy, excitotoxic injury or therapeutic responseto treatment thereto.

Alzheimer's disease, excitotoxic injury, neurogenesis, neurodegenerativediseases or therapeutic response to treatment thereto can be monitoredor diagnosed by measuring or detecting brain microtubule dynamics. Inthis method, the targeted molecules of interest are microtubules fromcentral or peripheral nervous system or microtubule subfractions (e.g.,tau-associated, dimeric, polymeric). In this format, the presentapplication is further directed to a method for monitoring or diagnosingAlzheimer's disease, excitotoxic injury, neurogenesis, neurodegenerativediseases or therapeutic response in a living system, the methodincluding: a) administering an isotope-labeled substrate to the livingsystem for a period of time sufficient for the isotope-labeled substrateto enter into the brain microtubule production pathway and thereby enterinto and label at least one microtubule from central or peripheralnervous system or one microtubule subfraction within the brainmicrotubule production pathway in the living system; b) obtaining one ormore samples from the living system, wherein the one or more samplesinclude at least one isotope-labeled microtubule from central orperipheral nervous system or one microtubule subfraction; c) measuringthe content, rate of incorporation and/or pattern or rate of change incontent and/or pattern of isotope labeling of the microtubules fromcentral or peripheral nervous system or microtubule subfractions; d)calculating molecular flux rates in the brain microtubule productionpathway based on the content and/or pattern or rate of change of contentand/or pattern of isotopic labeling in the microtubules from central orperipheral nervous system or microtubule subfractions to monitor ordiagnose Alzheimer's disease, excitotoxic injury, neurogenesis,neurodegenerative diseases or therapeutic response to treatment thereto.

Hepatic necrosis, toxin exposure, hepatitis or response to treatmentthereof can be monitored or diagnosed by measuring or detectinghepatocyte dynamics. In this method, the targeted molecule of interestis DNA from hepatocytes. In this format, the present application isfurther directed to a method for monitoring or diagnosing hepaticnecrosis, toxin exposure, hepatitis or response to treatment in a livingsystem, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the hepatocyte productionpathway and thereby enter into and label at least one DNA molecule fromhepatocytes within the hepatocyte production pathway in the livingsystem; b) obtaining one or more samples from the living system, whereinthe one or more samples include at least one isotope-labeled DNAmolecule from hepatocytes; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the DNA from hepatocytes; d) calculatingmolecular flux rates in the hepatocyte production pathway based on thecontent and/or pattern or rate of change of content and/or pattern ofisotopic labeling in the DNA from hepatocytes to monitor or diagnosehepatic necrosis, toxin exposure, hepatitis or response to treatmentthereof.

Hepatic fibrosis, cirrhosis risk, prognosis, disease activity orresponse to treatment thereof can be monitored or diagnosed by measuringor detecting hepatic collagen dynamics. In this method, the targetedmolecule of interest is collagen from liver. In this format, the presentapplication is further directed to a method for monitoring or diagnosinghepatic fibrosis, cirrhosis risk, prognosis, disease activity orresponse to treatment in a living system, the method including: a)administering an isotope-labeled substrate to the living system for aperiod of time sufficient for the isotope-labeled substrate to enterinto the hepatic collagen production pathway and thereby enter into andlabel at least one collagen molecule from liver within the hepaticcollagen production pathway in the living system; b) obtaining one ormore samples from the living system, wherein the one or more samplesinclude at least one isotope-labeled collagen molecule from liver; c)measuring the content, rate of incorporation and/or pattern or rate ofchange in content and/or pattern of isotope labeling of the collagenfrom liver; d) calculating molecular flux rates in the hepatic collagenproduction pathway based on the content and/or pattern or rate of changeof content and/or pattern of isotopic labeling in the collagen fromliver to monitor or diagnose hepatic fibrosis, cirrhosis risk,prognosis, disease activity or response to treatment thereof.

Effects from exposure to hepatic toxins, mitochondrial toxins, recoveryor response to treatment can be monitored or diagnosed by measuring ordetecting hepatic mitochondrial dynamics. In this method, the targetedmolecules of interest are DNA or phospholipids from hepaticmitochondria. In this format, the present application is furtherdirected to a method for monitoring or diagnosing effects from exposureto hepatic toxins, mitochondrial toxins, recovery or response totreatment in a living system, the method including: a) administering anisotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into the hepaticmitochondrial production pathway and thereby enter into and label atleast one DNA molecule or phospholipid from hepatic mitochondria withinthe hepatic mitochondrial production pathway in the living system; b)obtaining one or more samples from the living system, wherein the one ormore samples include at least one isotope-labeled DNA molecule orphospholipid from hepatic mitochondria; c) measuring the content, rateof incorporation and/or pattern or rate of change in content and/orpattern of isotope labeling of the DNA or phospholipids from hepaticmitochondria; d) calculating molecular flux rates in the hepaticmitochondrial production pathway based on the content and/or pattern orrate of change of content and/or pattern of isotopic labeling in the DNAor phospholipids from hepatic mitochondria to monitor or diagnoseeffects from exposure to hepatic toxins, mitochondrial toxins, recoveryor response to treatment.

Effects from exposure to nephrotoxins, recovery or response to treatmentcan be monitored or diagnosed by measuring or detecting renal epithelialcell dynamics. In this method, the targeted molecule of interest is DNAfrom renal epithelial cells. In this format, the present application isfurther directed to a method for monitoring or diagnosing effects fromexposure to nephrotoxins, recovery or response to treatment in a livingsystem, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the renal epithelial cellproduction pathway and thereby enter into and label at least one DNAmolecule from renal epithelial cells within the renal epithelial cellproduction pathway in the living system; b) obtaining one or moresamples from the living system, wherein the one or more samples includeat least one isotope-labeled DNA molecule from renal epithelial cells;c) measuring the content, rate of incorporation and/or pattern or rateof change in content and/or pattern of isotope labeling of the DNA fromrenal epithelial cells; d) calculating molecular flux rates in the renalepithelial cell production pathway based on the content and/or patternor rate of change of content and/or pattern of isotopic labeling in theDNA from renal epithelial cells to monitor or diagnose effects fromexposure to nephrotoxins, recovery or response to treatment.

Diabetes Mellitus nephropathy risk and activity or response to treatmentcan be monitored or diagnosed by measuring or detecting renal collagendynamics. In this method, the targeted molecule of interest is collagenfrom kidney. In this format, the present application is further directedto a method for monitoring or diagnosing DM nephropathy risk andactivity or response to treatment in a living system, the methodincluding: a) administering an isotope-labeled substrate to the livingsystem for a period of time sufficient for the isotope-labeled substrateto enter into the renal collagen production pathway and thereby enterinto and label at least one collagen molecule from kidney within therenal collagen production pathway in the living system; b) obtaining oneor more samples from the living system, wherein the one or more samplesinclude at least one isotope-labeled collagen molecule from kidney; c)measuring the content, rate of incorporation and/or pattern or rate ofchange in content and/or pattern of isotope labeling of the collagenfrom kidney; d) calculating molecular flux rates in the renal collagenproduction pathway based on the content and/or pattern or rate of changeof content and/or pattern of isotopic labeling in the collagen fromkidney to monitor or diagnose Diabetes Mellitus nephropathy risk andactivity or response to treatment.

Pulmonary fibrosis disease activity, black lung, hypersensitivitypneumonitis, asbestosis, silicosis or chronic obstructive pulmonarydisease or response to treatment thereof can be monitored or diagnosedby measuring or detecting pulmonary collagen dynamics. In this method,the targeted molecule of interest is pulmonary collagen. In this format,the present application is further directed to a method for monitoringor diagnosing pulmonary fibrosis disease activity, response totreatment; black lung, hypersensitivity pneumonitis, asbestosis,silicosis or chronic obstructive pulmonary disease in a living system,the method including: a) administering an isotope-labeled substrate tothe living system for a period of time sufficient for theisotope-labeled substrate to enter into the pulmonary collagenproduction pathway and thereby enter into and label at least onepulmonary collagen molecule within the pulmonary collagen productionpathway in the living system; b) obtaining one or more samples from theliving system, wherein the one or more samples include at least oneisotope-labeled pulmonary collagen molecule; c) measuring the content,rate of incorporation and/or pattern or rate of change in content and/orpattern of isotope labeling of the pulmonary collagen; d) calculatingmolecular flux rates in the pulmonary collagen production pathway basedon the content and/or pattern or rate of change of content and/orpattern of isotopic labeling in the pulmonary collagen to monitor ordiagnose pulmonary fibrosis disease activity, black lung,hypersensitivity pneumonitis, asbestosis, silicosis or chronicobstructive pulmonary disease or monitor response to treatment thereof.

Emphysema prognosis or therapeutic response to treatment thereto can bemonitored or diagnosed by measuring or detecting pulmonary elastindynamics. In this method, the targeted molecule of interest is pulmonaryelastin. In this format, the present application is further directed toa method for monitoring or diagnosing Emphysema prognosis or therapeuticresponse in a living system, the method including: a) administering anisotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into the pulmonaryelastin production pathway and thereby enter into and label at least onepulmonary elastin molecule within the pulmonary elastin productionpathway in the living system; b) obtaining one or more samples from theliving system, wherein the one or more samples include at least oneisotope-labeled pulmonary elastin molecule; c) measuring the content,rate of incorporation and/or pattern or rate of change in content and/orpattern of isotope labeling of the pulmonary elastin; d) calculatingmolecular flux rates in the pulmonary elastin production pathway basedon the content and/or pattern or rate of change of content and/orpattern of isotopic labeling in the pulmonary elastin to monitor ordiagnose emphysema prognosis or therapeutic response to treatmentthereto.

Inflammatory bowel disease activity, prognosis or therapeutic responseto treatment thereto can be monitored or diagnosed by measuring ordetecting colonocyte DNA dynamics. In this method, the targeted moleculeof interest is DNA from colonocytes isolated from stool, colon biopsy,or other colon tissue sample. In this format, the present application isfurther directed to a method for monitoring or diagnosing inflammatorybowel disease activity, prognosis or therapeutic response in a livingsystem, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the colonocyte DNA replicationpathway and thereby enter into and label at least one DNA molecule fromcolonocytes within the colonocyte DNA replication pathway in the livingsystem; b) obtaining one or more samples from the living system, whereinthe one or more samples include at least one isotope-labeled DNAmolecule from colonocytes; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the DNA from colonocytes; d) calculatingmolecular flux rates in the colonocyte DNA replication pathway based onthe content and/or pattern or rate of change of content and/or patternof isotopic labeling in the DNA from colonocytes to monitor or diagnoseinflammatory bowel disease activity, prognosis or therapeutic responseto treatment thereto.

H. pylori activity or therapeutic response to treatment thereto, cancerrisk or gastric cancer can be monitored or diagnosed by measuring ordetecting gastric epithelial DNA dynamics. In this method, the targetedmolecule of interest is DNA from gastric epithelial cells. In thisformat, the present application is further directed to a method formonitoring or diagnosing H. pylori activity or therapeutic response,cancer risk or gastric cancer in a living system, the method including:a) administering an isotope-labeled substrate to the living system for aperiod of time sufficient for the isotope-labeled substrate to enterinto the gastric epithelial DNA replication pathway and thereby enterinto and label at least one DNA molecule from gastric epithelial cellswithin the gastric epithelial DNA replication pathway in the livingsystem; b) obtaining one or more samples from the living system, whereinthe one or more samples include at least one isotope-labeled DNAmolecule from gastric epithelial cells; c) measuring the content, rateof incorporation and/or pattern or rate of change in content and/orpattern of isotope labeling of the DNA from gastric epithelial cells; d)calculating molecular flux rates in the gastric epithelial DNAreplication pathway based on the content and/or pattern or rate ofchange of content and/or pattern of isotopic labeling in the DNA fromgastric epithelial cells to monitor or diagnose H. pylori activity ortherapeutic response to treatment thereto, cancer risk or gastriccancer.

Cell mediated immunity, immune activation, AIDS or therapeutic responseto treatment thereto can be monitored or diagnosed by measuring ordetecting T-cell dynamics. In this method, the targeted molecule ofinterest is DNA from T-cells. In this format, the present application isfurther directed to a method for monitoring or diagnosing cell mediatedimmunity, immune activation, AIDS or therapeutic response in a livingsystem, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the T-cell production pathwayand thereby enter into and label at least one DNA molecule from T-cellswithin the T-cell production pathway in the living system; b) obtainingone or more samples from the living system, wherein the one or moresamples include at least one isotope-labeled DNA molecule from T-cells;c) measuring the content, rate of incorporation and/or pattern or rateof change in content and/or pattern of isotope labeling of the DNA fromT-cells; d) calculating molecular flux rates in the T-cell productionpathway based on the content and/or pattern or rate of change of contentand/or pattern of isotopic labeling in the DNA from T-cells to monitoror diagnose cell mediated immunity, immune activation, AIDS ortherapeutic response to treatment thereto.

Vaccination response can be monitored or diagnosed by measuring ordetecting antigen-specific T-cell dynamics. In this method, the targetedmolecule of interest is DNA from T-cells isolated based on their antigenspecificity. In this format, the present application is further directedto a method for monitoring or diagnosing Y in a living system, themethod including: a) administering an isotope-labeled substrate to theliving system for a period of time sufficient for the isotope-labeledsubstrate to enter into the antigen-specific T-cell production pathwayand thereby enter into and label at least one DNA molecule from T-cellsisolated based on their antigen specificity within the antigen-specificT-cell production pathway in the living system; b) obtaining one or moresamples from the living system, wherein the one or more samples includeat least one isotope-labeled DNA molecule from T-cells isolated based ontheir antigen specificity; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the DNA from T-cells isolated based on theirantigen specificity; d) calculating molecular flux rates in theantigen-specific T-cell production pathway based on the content and/orpattern or rate of change of content and/or pattern of isotopic labelingin the DNA from T-cells isolated based on their antigen specificity tomonitor vaccination response.

Thymopoiesis, thymic failure or therapeutic response to treatmentthereto can be monitored or diagnosed by measuring or detecting naïveT-cell dynamics. In this method, the targeted molecule of interest isDNA from naïve T-cells. In this format, the present application isfurther directed to a method for monitoring or diagnosing thymopoiesis,thymic failure or therapeutic response in a living system, the methodincluding: a) administering an isotope-labeled substrate to the livingsystem for a period of time sufficient for the isotope-labeled substrateto enter into the naïve T-cell production pathway and thereby enter intoand label at least one DNA molecule from naïve T-cells within the naïveT-cell production pathway in the living system; b) obtaining one or moresamples from the living system, wherein the one or more samples includeat least one isotope-labeled DNA molecule from naïve T-cells; c)measuring the content, rate of incorporation and/or pattern or rate ofchange in content and/or pattern of isotope labeling of the DNA fromnaïve T-cells; d) calculating molecular flux rates in the naïve T-cellproduction pathway based on the content and/or pattern or rate of changeof content and/or pattern of isotopic labeling in the DNA from naïveT-cells to monitor or diagnose thymopoiesis, thymic failure ortherapeutic response to treatment thereto.

B-cell/plasma cell activity, or therapeutic response to treatmentthereto or vaccine response can be monitored or diagnosed by measuringor detecting specific antibody dynamics. In this method, the targetedmolecule of interest is the antibody specific to the antigen of choice.In this format, the present application is further directed to a methodfor monitoring or diagnosing B-cell/plasma cell activity, compound ortherapeutic response or vaccine response in a living system, the methodincluding: a) administering an isotope-labeled substrate to the livingsystem for a period of time sufficient for the isotope-labeled substrateto enter into the specific antibody production pathway and thereby enterinto and label at least one antibody specific to the antigen of choicewithin the specific antibody production pathway in the living system; b)obtaining one or more samples from the living system, wherein the one ormore samples include at least one isotope-labeled antibody specific tothe antigen of choice; c) measuring the content, rate of incorporationand/or pattern or rate of change in content and/or pattern of isotopelabeling of the antibody specific to the antigen of choice; d)calculating molecular flux rates in the specific antibody productionpathway based on the content and/or pattern or rate of change of contentand/or pattern of isotopic labeling in the antibody specific to theantigen of choice to monitor or diagnose B-cell/plasma cell activity, ortherapeutic response or vaccine response.

Immune activation or disease activity can be monitored or diagnosed bymeasuring or detecting serum acute-phase reactant dynamics. In thismethod, the targeted molecules of interest are acute phase proteins. Inthis format, the present application is further directed to a method formonitoring or diagnosing immune activation or disease activity in aliving system, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the serum acute-phase reactantproduction pathway and thereby enter into and label at least one acutephase protein within the serum acute-phase reactant production pathwayin the living system; b) obtaining one or more samples from the livingsystem, wherein the one or more samples include at least oneisotope-labeled acute phase protein; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the acute phase proteins; d) calculatingmolecular flux rates in the serum acute-phase reactant productionpathway based on the content and/or pattern or rate of change of contentand/or pattern of isotopic labeling in the acute phase proteins tomonitor or diagnose immune activation or disease activity.

Humoral immunity can be monitored or diagnosed by measuring or detectingplasma cell dynamics. In this method, the targeted molecule of interestis DNA from plasma cells. In this format, the present application isfurther directed to a method for monitoring or diagnosing humoralimmunity in a living system, the method including: a) administering anisotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into the plasmacell production pathway and thereby enter into and label at least oneDNA molecule from plasma cells within the plasma cell production pathwayin the living system; b) obtaining one or more samples from the livingsystem, wherein the one or more samples include at least oneisotope-labeled DNA molecule from plasma cells; c) measuring thecontent, rate of incorporation and/or pattern or rate of change incontent and/or pattern of isotope labeling of the DNA from plasma cells;d) calculating molecular flux rates in the plasma cell productionpathway based on the content and/or pattern or rate of change of contentand/or pattern of isotopic labeling in the DNA from plasma cells tomonitor or diagnose humoral immunity.

Host defense activity or therapeutic response (e.g., IL-2) can bemonitored or diagnosed by measuring or detecting natural killer celldynamics. In this method, the targeted molecule of interest is DNA fromnatural killer cells. In this format, the present application is furtherdirected to a method for monitoring or diagnosing host defense activityor therapeutic response in a living system, the method including: a)administering an isotope-labeled substrate to the living system for aperiod of time sufficient for the isotope-labeled substrate to enterinto the natural killer cell production pathway and thereby enter intoand label at least one DNA molecule from natural killer cells within thenatural killer cell production pathway in the living system; b)obtaining one or more samples from the living system, wherein the one ormore samples include at least one isotope-labeled DNA molecule fromnatural killer cells; c) measuring the content, rate of incorporationand/or pattern or rate of change in content and/or pattern of isotopelabeling of the DNA from natural killer cells; d) calculating molecularflux rates in the natural killer cell production pathway based on thecontent and/or pattern or rate of change of content and/or pattern ofisotopic labeling in the DNA from natural killer cells to monitor ordiagnose host defense activity or therapeutic response.

Endogenous response to exogenous compound or therapeutic or host defensecan be monitored or diagnosed by measuring or detecting cytokinedynamics. In this method, the targeted molecules of interest aresecreted or tissue associated cytokines. In this format, the presentapplication is further directed to a method for monitoring or diagnosingendogenous response to exogenous compound or therapeutic or host defensein a living system, the method including: a) administering anisotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into the cytokineproduction pathway and thereby enter into and label at least onesecreted or tissue associated cytokine within the cytokine productionpathway in the living system; b) obtaining one or more samples from theliving system, wherein the one or more samples include at least oneisotope-labeled secreted or tissue associated cytokine; c) measuring thecontent, rate of incorporation and/or pattern or rate of change incontent and/or pattern of isotope labeling of the secreted or tissueassociated cytokines; d) calculating molecular flux rates in thecytokine production pathway based on the content and/or pattern or rateof change of content and/or pattern of isotopic labeling in the secretedor tissue associated cytokines to monitor or diagnose endogenousresponse to exogenous compound or therapeutic or host defense.

Viral replication, disease activity, or therapeutic response orsensitivity to antiviral agents can be monitored or diagnosed bymeasuring or detecting viral DNA/RNA dynamics (e.g., HIV, Hepatitis B).In this method, the targeted molecule of interest is DNA or RNA from thevirus of interest. In this format, the present application is furtherdirected to a method for monitoring or diagnosing viral replication,disease activity, compound or therapeutic response or sensitivity toantiviral agents in a living system, the method including: a)administering an isotope-labeled substrate to the living system for aperiod of time sufficient for the isotope-labeled substrate to enterinto the viral DNA/RNA synthesis pathways and thereby enter into andlabel at least one DNA or RNA molecule from the virus of interest withinthe viral DNA/RNA synthesis pathways in the living system; b) obtainingone or more samples from the living system, wherein the one or moresamples include at least one isotope-labeled DNA or RNA molecule fromthe virus of interest; c) measuring the content, rate of incorporationand/or pattern or rate of change in content and/or pattern of isotopelabeling of the DNA or RNA from the virus of interest; d) calculatingmolecular flux rates in the viral DNA/RNA synthesis pathways based onthe content and/or pattern or rate of change of content and/or patternof isotopic labeling in the DNA or RNA from the virus of interest tomonitor or diagnose viral replication, disease activity, or monitortherapeutic response or sensitivity to antiviral agents.

Viral replication, disease activity, or therapeutic response orsensitivity to antiviral agents can be monitored or diagnosed bymeasuring or detecting viral protein dynamics. In this method, thetargeted molecule of interest is protein from the virus of interest. Inthis format, the present application is further directed to a method formonitoring or diagnosing viral replication, disease activity, compoundor therapeutic response or sensitivity to antiviral agents in a livingsystem, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the viral protein synthesispathway and thereby enter into and label at least one protein from thevirus of interest within the viral protein synthesis pathway in theliving system; b) obtaining one or more samples from the living system,wherein the one or more samples include at least one isotope-labeledprotein from the virus of interest; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the protein from the virus of interest; d)calculating molecular flux rates in the viral protein synthesis pathwaybased on the content and/or pattern or rate of change of content and/orpattern of isotopic labeling in the protein from the virus of interestto monitor or diagnose viral replication, disease activity, or monitortherapeutic response or sensitivity to antiviral agents.

Bacterial cell division, disease activity or response to antibiotics canbe monitored or diagnosed by measuring or detecting bacterial dynamics.In this method, the targeted molecule of interest is DNA or othermolecule (e.g., protein, carbohydrate, lipid) from the bacteria ofinterest. In this format, the present application is further directed toa method for monitoring or diagnosing bacterial cell division, diseaseactivity or response to antibiotics in a living system, the methodincluding: a) administering an isotope-labeled substrate to the livingsystem for a period of time sufficient for the isotope-labeled substrateto enter into the bacterial replication pathway and thereby enter intoand label at least one DNA or other molecule from the bacteria ofinterest within the bacterial replication pathway in the living system;b) obtaining one or more samples from the living system, wherein the oneor more samples include at least one isotope-labeled DNA or othermolecule from the bacteria of interest; c) measuring the content, rateof incorporation and/or pattern or rate of change in content and/orpattern of isotope labeling of the DNA or other molecule from thebacteria of interest; d) calculating molecular flux rates in thebacterial replication pathway based on the content and/or pattern orrate of change of content and/or pattern of isotopic labeling in the DNAor other molecule from the bacteria of interest to monitor or diagnosebacterial cell division, disease activity or response to antibiotics.

Parasite division and growth or therapeutic response (e.g., malaria,schistosomiasis) can be monitored or diagnosed by measuring or detectingparasite dynamics. In this method, the targeted molecule of interest isDNA or other molecule (e.g., protein, carbohydrate, lipid) from theparasite of interest. In this format, the present application is furtherdirected to a method for monitoring or diagnosing parasite division andgrowth or therapeutic response in a living system, the method including:a) administering an isotope-labeled substrate to the living system for aperiod of time sufficient for the isotope-labeled substrate to enterinto the parasite replication pathway and thereby enter into and labelat least one DNA or other molecule from the parasite of interest withinthe parasite replication pathway in the living system; b) obtaining oneor more samples from the living system, wherein the one or more samplesinclude at least one isotope-labeled DNA or other molecule from theparasite of interest; c) measuring the content, rate of incorporationand/or pattern or rate of change in content and/or pattern of isotopelabeling of the DNA or other molecule from the parasite of interest; d)calculating molecular flux rates in the parasite replication pathwaybased on the content and/or pattern or rate of change of content and/orpattern of isotopic labeling in the DNA or other molecule from theparasite of interest to monitor or diagnose parasite division and growthor therapeutic response.

Infectious activity or therapeutic response to treatment thereof can bemonitored or diagnosed by measuring or detecting intestinal microbialdynamics. In this method, the targeted molecule of interest is DNA orother molecule (e.g., protein, carbohydrate, lipid) from intestinalbacteria. In this format, the present application is further directed toa method for monitoring or diagnosing infectious activity or therapeuticresponse in a living system, the method including: a) administering anisotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into theintestinal microbial replication pathway and thereby enter into andlabel at least one DNA or other molecule from intestinal bacteria withinthe intestinal microbial replication pathway in the living system; b)obtaining one or more samples from the living system, wherein the one ormore samples include at least one isotope-labeled DNA or other moleculefrom intestinal bacteria; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the DNA or other molecule from intestinalbacteria; d) calculating molecular flux rates in the intestinalmicrobial replication pathway based on the content and/or pattern orrate of change of content and/or pattern of isotopic labeling in the DNAor other molecule from intestinal bacteria to monitor or diagnoseinfectious activity or therapeutic response to treatment thereof.

Abscess, empyema or therapeutic response to treatment thereof can bemonitored or diagnosed by measuring or detecting bacterial dynamics in aclosed space. In this method, the targeted molecule of interest isbacterial DNA or other molecule (e.g., protein, carbohydrate, lipid)from tissue or abscess or fluid sample. In this format, the presentapplication is further directed to a method for monitoring or diagnosingabscess, empyema or therapeutic response in a living system, the methodincluding: a) administering an isotope-labeled substrate to the livingsystem for a period of time sufficient for the isotope-labeled substrateto enter into the bacterial replication pathway and thereby enter intoand label at least one bacterial DNA or other molecule from tissue orabscess or fluid sample within the bacterial replication pathway in theliving system; b) obtaining one or more samples from the living system,wherein the one or more samples include at least one isotope-labeledbacterial DNA or other molecule from tissue or abscess or fluid sample;c) measuring the content, rate of incorporation and/or pattern or rateof change in content and/or pattern of isotope labeling of the bacterialDNA or other molecule from tissue or abscess or fluid sample; d)calculating molecular flux rates in the bacterial replication pathwaybased on the content and/or pattern or rate of change of content and/orpattern of isotopic labeling in the bacterial DNA or other molecule fromtissue or abscess or fluid sample to monitor or diagnose abscess,empyema or therapeutic response to treatment thereto.

Endocarditis or therapeutic response to treatment thereto can bemonitored or diagnosed by measuring or detecting endovascular bacterialdynamics. In this method, the targeted molecule of interest is DNA orother molecule (e.g., protein, carbohydrate, lipid) from endovascularbacteria. In this format, the present application is further directed toa method for monitoring or diagnosing endocarditis or therapeuticresponse to treatment thereto in a living system, the method including:a) administering an isotope-labeled substrate to the living system for aperiod of time sufficient for the isotope-labeled substrate to enterinto the endovascular bacterial replication pathway and thereby enterinto and label at least one DNA or other molecule from endovascularbacteria within the endovascular bacterial replication pathway in theliving system; b) obtaining one or more samples from the living system,wherein the one or more samples include at least one isotope-labeled DNAor other molecule from endovascular bacteria; c) measuring the content,rate of incorporation and/or pattern or rate of change in content and/orpattern of isotope labeling of the DNA or other molecule fromendovascular bacteria; d) calculating molecular flux rates in theendovascular bacterial replication pathway based on the content and/orpattern or rate of change of content and/or pattern of isotopic labelingin the DNA or other molecule from endovascular bacteria to monitor ordiagnose endocarditis or therapeutic response to treatment thereto.

Stem cell response (transplant, compound or therapeutic) or status ofcytopenias can be monitored or diagnosed by measuring or detecting bonemarrow precursor/marrow cell dynamics. In this method, the targetedmolecule of interest is DNA from bone marrow precursor/marrow cells. Inthis format, the present application is further directed to a method formonitoring or diagnosing stem cell response or status of cytopenias in aliving system, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the bone marrow precursor/marrowcell production pathway and thereby enter into and label at least oneDNA molecule from bone marrow precursor/marrow cells within the bonemarrow precursor/marrow cell production pathway in the living system; b)obtaining one or more samples from the living system, wherein the one ormore samples include at least one isotope-labeled DNA molecule from bonemarrow precursor/marrow cells; c) measuring the content, rate ofincorporation and/or pattern or rate of change in content and/or patternof isotope labeling of the DNA from bone marrow precursor/marrow cells;d) calculating molecular flux rates in the bone marrow precursor/marrowcell production pathway based on the content and/or pattern or rate ofchange of content and/or pattern of isotopic labeling in the DNA frombone marrow precursor/marrow cells to monitor or diagnose stem cellresponse or status of cytopenias.

Hemolysis, anemia response (reticulocytosis), hemoglobinopathies ortreatment thereof can be monitored or diagnosed by measuring ordetecting hemoglobin dynamics in red blood cells. In this method, thetargeted molecule of interest is hemoglobin. In this format, the presentapplication is further directed to a method for monitoring or diagnosinghemolysis, anemia response (reticulocytosis) or hemoglobinopathies in aliving system, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the hemoglobin synthesis pathwayand thereby enter into and label at least one hemoglobin molecule withinthe hemoglobin synthesis pathway in the living system; b) obtaining oneor more samples from the living system, wherein the one or more samplesinclude at least one isotope-labeled hemoglobin molecule; c) measuringthe content, rate of incorporation and/or pattern or rate of change incontent and/or pattern of isotope labeling of the hemoglobin; d)calculating molecular flux rates in the hemoglobin synthesis pathwaybased on the content and/or pattern or rate of change of content and/orpattern of isotopic labeling in the hemoglobin to monitor or diagnosehemolysis, anemia response (reticulocytosis), hemoglobinopathies ortreatment thereof.

Thrombocytopenia, thrombocytosis or treatment thereof can be monitoredor diagnosed by measuring or detecting platelet phospholipid dynamics.In this method, the targeted molecule of interest is one or morephospholipids or DNA from platelets or platelet precursors. In thisformat, the present application is further directed to a method formonitoring or diagnosing thrombocytopenia or thrombocytosis in a livingsystem, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the platelet phospholipidsynthesis pathway and thereby enter into and label at least oneisotope-labeled phospholipid or DNA molecule from platelets or plateletprecursors within the platelet phospholipid synthesis pathway in theliving system; b) obtaining one or more samples from the living system,wherein the one or more samples include at least one isotope-labeledphospholipid or DNA molecule from platelets or platelet precursors; c)measuring the content, rate of incorporation and/or pattern or rate ofchange in content and/or pattern of isotope labeling of thephospholipids or DNA from platelets or platelet precursors; d)calculating molecular flux rates in the platelet phospholipid synthesispathway based on the content and/or pattern or rate of change of contentand/or pattern of isotopic labeling in the phospholipids or DNA fromplatelets or platelet precursors to monitor or diagnosethrombocytopenia, thrombocytosis or treatment thereof.

Anemia, hemolysis or therapeutic response to treatment thereto can bemonitored or diagnosed by measuring or detecting erythrocyte membranedynamics. In this method, the targeted molecule of interest isphospholipid from erythrocytes. In this format, the present applicationis further directed to a method for monitoring or diagnosing anemia,hemolysis or therapeutic response to treatment thereto in a livingsystem, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the erythrocyte membraneproduction pathway and thereby enter into and label at least onephospholipid from erythrocytes within the erythrocyte membraneproduction pathway in the living system; b) obtaining one or moresamples from the living system, wherein the one or more samples includeat least one isotope-labeled phospholipid from erythrocytes; c)measuring the content, rate of incorporation and/or pattern or rate ofchange in content and/or pattern of isotope labeling of the phospholipidfrom erythrocytes; d) calculating molecular flux rates in theerythrocyte membrane production pathway based on the content and/orpattern or rate of change of content and/or pattern of isotopic labelingin the phospholipid from erythrocytes to monitor or diagnose anemia,hemolysis or therapeutic response to treatment thereto.

Spermatogenesis, male infertility, or therapeutic response to treatmentthereto or endocrine disruptors can be monitored or diagnosed bymeasuring or detecting spermatocyte dynamics. In this method, thetargeted molecule of interest is DNA from spermatocytes. In this format,the present application is further directed to a method for monitoringor diagnosing spermatogenesis, male infertility, compound or therapeuticresponse to treatment thereto or endocrine disruptors in a livingsystem, the method including: a) administering an isotope-labeledsubstrate to the living system for a period of time sufficient for theisotope-labeled substrate to enter into the spermatogenesis pathway andthereby enter into and label at least one DNA molecule fromspermatocytes within the spermatogenesis pathway in the living system;b) obtaining one or more samples from the living system, wherein the oneor more samples include at least one isotope-labeled DNA molecule fromspermatocytes; c) measuring the content, rate of incorporation and/orpattern or rate of change in content and/or pattern of isotope labelingof the DNA from spermatocytes; d) calculating molecular flux rates inthe spermatogenesis pathway based on the content and/or pattern or rateof change of content and/or pattern of isotopic labeling in the DNA fromspermatocytes to monitor or diagnose spermatogenesis, male infertility,or therapeutic response to treatment thereto or endocrine disruptors.

Developmental biology and disorders thereof can be monitored ordiagnosed by measuring or detecting the timing of embryonic protein andlipid dynamics. In this method, the targeted molecules of interest areembryonic proteins, lipids, or DNA. In this format, the presentapplication is further directed to a method for monitoring or diagnosingdevelopmental biology and disorders thereof in a living system, themethod including: a) administering an isotope-labeled substrate to theliving system for a period of time sufficient for the isotope-labeledsubstrate to enter into the embryonic protein and lipid productionpathways and thereby enter into and label at least one embryonicprotein, lipid, or DNA molecule within the embryonic protein and lipidproduction pathways in the living system; b) obtaining one or moresamples from the living system, wherein the one or more samples includeat least one isotope-labeled embryonic protein, lipid, or DNA molecule;c) measuring the content, rate of incorporation and/or pattern or rateof change in content and/or pattern of isotope labeling of the embryonicproteins, lipids, or DNA; d) calculating molecular flux rates in theembryonic protein and lipid production pathways based on the contentand/or pattern or rate of change of content and/or pattern of isotopiclabeling in the embryonic proteins, lipids, or DNA to monitor ordiagnose developmental biology and disorders thereof.

Genetic instability or cancer risk can be monitored or diagnosed bymeasuring or detecting genomic DNA dynamics. In this method, thetargeted molecule of interest is genomic DNA (from at risk tissue ifappropriate). In this format, the present application is furtherdirected to a method for monitoring or diagnosing genetic instability orcancer risk in a living system, the method including: a) administeringan isotope-labeled substrate to the living system for a period of timesufficient for the isotope-labeled substrate to enter into the genomicDNA replication pathway and thereby enter into and label at least onegenomic DNA molecule within the genomic DNA replication pathway in theliving system; b) obtaining one or more samples from the living system,wherein the one or more samples include at least one isotope-labeledgenomic DNA molecule; c) measuring the content, rate of incorporationand/or pattern or rate of change in content and/or pattern of isotopelabeling of the genomic DNA; d) calculating molecular flux rates in thegenomic DNA replication pathway based on the content and/or pattern orrate of change of content and/or pattern of isotopic labeling in thegenomic DNA to monitor or diagnose genetic instability or cancer risk.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the drug discovery, development,and approval (DDA) process using effects on biomarkers (i.e., datacollected by the methods of the present invention) as a means fordeciding to continue or cease efforts.

FIG. 2 shows a schematic diagram of an example metabolic pathway (DNAsynthesis, both de novo and salvage) and various component elements.Locations of stable or radioactive isotope labeling are shown.G6P=Glucose-6-phosphate. R5P=ribose-5-phosphate.PRPP=5-phosphoribosyl-α-pyrophospate. NDP=nucleotide diphosphate.dNTP=deoxynucleotide triphosphate. RR=ribonucleotide reductase.dN=deoxynucleotide. ³H-dT=tritiated deoxythymidine.BrdU=5-bromo-2-deoxyuridine. GNG=gluconeogenesis. DNNS=de novonucleotide synthesis. DNPS=de novo precursor synthesis.

FIG. 3 shows label (i.e., ²H from ²H₂O) being incorporated into DNA.

FIG. 4 shows label incorporation into collagen by way of labeled aminoacids.

FIG. 5 shows the synthesis and degradation pathways for collagen.

FIG. 6 illustrates use of the inventions herein in a drug discoveryprocess.

FIG. 7 depicts the effects of chronic imipramine treatment onhippocampal neuroprogenitor cell proliferation in male 129SvEv mice.Chronic Imipramine treatment increases progenitor cell proliferation inmale 129SvEv mice. Mice received 3 weeks of treatment with imipramine(20 mg/kg/day) in drinking water and were labeled with 10% ²H₂O duringthe last 3 days of treatment. Data represent mean±SD, n=4 per group.

FIG. 8 depicts the effects of chronic fluoxetine treatment onhippocampal neuroprogenitor cell proliferation in male 129SvEv mice.Fluoxetine increases progenitor cell proliferation. Mice received 2weeks of treatment with fluoxetine (10 mg/kg/day) in drinking water andwere labeled with 10% ²H₂O during the last week of treatment. Datarepresent mean±SD, n=5 per group, *p<0.05 significantly different fromvehicle group.

FIG. 9 depicts increased islet (beta-cell) proliferation after 50%pancreatectomy in Wistar rats.

FIG. 10 depicts the effects of two demethylating drugs (azacitidine anddecitabine) on SW 1753 cells that were cultured overnight in DMEM media(10% FBS) supplemented with 20 mM ²H3-methyl methionine. Three differentconcentrations (125 nM, 250 nM, and 500 nM) of azacitidine anddecitabine were then added using methyldeoxycytidine (125 nM and 500 nM)as a negative control. Decitabine showed a better efficacy in inhibitingmethylation (70% reduction) at 250 nM when compared to azacytidine (lessthan 10%).

FIG. 11 depicts spermatocyte labeling curves for eleven subjects withnormal semen analyses. Men were labeled with 50 ml of 70% ²H₂O twicedaily for 3 weeks. Semen samples were collected every two weeks for 90days from the start on ²H₂O, Spermatocyte DNA enrichment was measured byGC/MS and compared to that of a fully-turned over cell (monocyte) tocalculate the percentage of new cells present.

FIG. 12 depicts turnover of cerebral cortical tissue in response tobacterial toxin lipopolysaccharide (LPS). The high dose of LPS affectedcortical tissue turnover representing the response of the brain to toxicinsult.

FIG. 13 depicts in vivo dose response of liver cell proliferation over 7days of carbon tetrachloride treatment. Swiss Webster mice were given IPinjections of CCl₄ over 7 days concurrent with ²H₂O.

FIG. 14 depicts microglial response to the neuroinflammatory toxinlipopolysachamide (LPS). LPS is administered every other day to miceintraperitoneally at the two doses indicated (1 and 4 mg/kg bodyweight). ²H₂O was administered concurrently. Some animals were harvestedat each of the indicated time points, and microglia were isolated andanalyzed for deuterium incorporation. The increased proliferativeresponse to both doses is significant with respect to control at everytime point measured (p<0.05, ANOVA/Tukey), and dose dependence isobserved from day 14 forward (p<0.05, ANOVA/Tukey). Error bars indicatestandard deviation.

FIG. 15 depicts the effects of two anti-inflammatory chemotherapeuticagents (dexamethasone and minocycline) on microglial proliferation. Micewere given ²H₂O and either intraperitoneal lipopolysaccharide (LPS) toinduce neuroinflammation, or a vehicle (control). Mice given LPS weretreated with either a vehicle or dexamethasone or minocycline. Microgliawere then isolated and analyzed for deuterium incorporation as describedherein. The results show that the microglial proliferation assay iscapable of detecting the activity, in vivo, of an effectiveanti-neuroinflammatory therapeutic. The LPS treated groups showincreased proliferation with respect to the control group (p<0.01,ANOVA/Tukey) but the dexamethasone and minocycline treated groups showsuppressed proliferation with respect to the vehicle treated groups(p<0.001, ANOVA/Tukey).

FIG. 16 depicts keratin kinetics in normal and fsn mice. Fsn mice aremutant mice with a psoriasis-like phenotype. Keratin turnover isdramatically enhanced in fsn mice as measured by deuterium incorporationinto keratin—fsn mice reach maximal labeling in 4 days as opposed to 15for control animals.

FIG. 17 depicts keratinocyte kinetics in normal and fsn mice. Fsn miceare mutant mice with a psoriasis-like phenotype. Keratinocyte turnoveris enhanced in fsn mice as measured by deuterium incorporation intokeratinocyte DNA—fsn mice reach maximal labeling in 4 days as opposed 30for control animals.

FIG. 18 depicts proliferation of endothelial cells from xenograft tumorsin mice treated with an anti-angiogenic drug (Avastin) or a vehicle(saline). Endothelial cell proliferation is suppressed in animalstreated with the drug.

FIG. 19 depicts the effect of therapeutic agents on carbon tetrachloride(CCl₄)-induced liver fibrosis. Both Interferon-gamma (a) androsiglitazone (b) reduce the rate of collagen synthesis in mice treatedwith CCl₄.

FIG. 20 depicts in vivo tumor cell proliferation over 5 days ofchemotherapy treatment. Female balb/C mice were implanted subcutaneouslywith approximately 106 EMT7 mouse mammary carcinoma cells in matrigeland allowed to reach ca. 1500 mm³. Mice were labeled with ²H₂O with ani.p. bolus followed by 8% ²H₂O in drinking water for the duration of thestudy. Mice received concurrent treatment with either 125 mg/kg Gem or500 mg/kg HU. Gem was administered every other day, HU daily. At the endof 5 days tumors were removed, homogenized and DNA was isolated asdescribed in Example 2, infra. Both chemotherapeutic agents suppressedtumor cell and bone marrow proliferation.

FIG. 21 depicts lipolysis (a) and adipose tissue TG synthesis (b) inmice. Lipolysis and adipose tissue TG synthesis were measured in normalmice, untreated ob/ob mice, ob/ob mice pair fed against control mice,and ob/ob mice treated with leptin. Leptin-treated mice showed asignificant decrease in TG synthesis over the course of the study.Abbreviations: TG=triglyceride; g=grams.

FIG. 22 depicts the response of different bone marrow cell subsets totreatment with hydroxyurea (OHU). Total bone marrow (TBM) was analyzed,or cells were divided into lymphoid, myeloid, or other cells, andanalyzed separately. In three cases (indicated by **) OHU suppressedbone marrow proliferation.

FIG. 23 depicts the response of different bone marrow cell subsets totreatment with interleukin-1 (IL-1) after OHU-mediated myelosuppression.TBM is total bone marrow, or cells were divided into lymphoid, myeloid,or other cells, and analyzed separately. In three cases (indicated by**) IL-1 is capable of stimulating myeloid cell proliferation. Theeffect in lymphoid cells is not statistically significant.

FIG. 24 depicts rates of glycolytic disposal as determined by measuringthe production of deuterated water after administration of deuteratedglucose. Blood was collected and analyzed 60 and 90 minutes afteradministration of deuterated glucose. SD controls represent normalanimals, ZDF animals are a model of pre-diabetes, and show decreasedglycolytic disposal. After treatment, ZDF animals have a glycolyticdisposal rate that is similar to healthy control animals. This datashows a clear effect of a known insulin-sensitizing drug (rosiglitazone)in an animal model of disease.

FIG. 25 depicts serum protein synthesis rates (calculated using totalprotein concentration and deuterium enrichment rates for each protein)in a normal volunteer or a myeloma patient. In the myeloma patient, thesynthesis rates of the serum proteins evaluated are all suppressed infavor of the M-protein, which is produced by malignant cells. A kineticanalysis of the type described is very sensitive to such changes insynthesis.

FIG. 26 depicts the fractional synthesis of myelin as determined bymeasuring deuterium incorporation into galactocerebroside (GalCer).Animals were treated as described for either 6 or 9 weeks, and labeledwith deuterated water for the last 3 weeks of treatment. Control animals(closed diamonds) show a synthesis rate of 12% new (GalCer) in 3 weeks.Animals treated with a demyelinating toxin, cuprizone, show a decreasein the rate of fractional synthesis—reduced to about 5% new (closedsquares). Animals treated with cuprizone for 6 weeks, and then givendeuterated water for 3 weeks beginning at the time cuprizone treatmentceased show a dramatic increase in fractional synthesis (open triangles)as remyelination occurs.

FIG. 27 depicts the proliferation of mature neurons in normal andantidepressant (imipramine)-treated adult mice. Neurons were isolated byflow cytometry and analyzed as described in Example 2, infra.Imipramine, a known antidepressant, increased the rate of mature neuronformation.

FIG. 28 depicts pancreatic islet/beta cell proliferation in a rat modelof pre-diabetes (Zucker fat), a rat model of diabetes (Zucker-diabetes)and control animals (SD-control). Diabetic rats have impaired islet/betacell proliferation, as expected from a diabetic animal. Pre-diabeticanimals show increased proliferation of islet/beta cells, as thepancreas responds to decreasing insulin sensitivity.

FIG. 29 depicts the effect of a high fat diet on normal rats as measuredby the glycolytic disposal test. Deuterated water produced as apercentage of the total possible from the administered glucose load (%load) is shown for normal and high-fat diet (3 weeks) rats. Data wascollected 60 and 90 minutes after administration of deuterated glucose.

FIG. 30 depicts whole body glycolytic disposal in human patients with avariety of conditions. Lean=lean normal subjects. Overweight=overweightsubjects. Obese=obese subjects. DM2=diabetes type II. HIV+=HIV positivepatients. Results are presented as moles of ²H₂O produced per kg of leanbody mass. Measurements were made 4 hours after the administration of a15 gram glucose load.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

In biochemical terms, the processes that underlie most diseases can bestbe described as molecular fluxes through complex biochemical pathways ornetworks. Biochemical pathways include linked series of biochemical,physical or spatial transformations typically catalyzed by proteins andoccurring in vivo in the context of highly complex networks. Theproteins that catalyze the flow of molecules through pathways are codedby genes. Contemporary drug discovery, development and approval (DDA)has therefore largely consisted of strategies for identifying andmodulating individual proteins or genes that comprise the elementalcomponents of the pathways that are believed to be critically involvedin a disease. Modern drug research tools include gene expressionprofiling, proteomics, high throughput screening of enzyme activities,and combinatorial chemistry libraries for screening against specificprotein targets. Such genes and proteins are termed potentialtherapeutic targets in contemporary drug research and compounds that mayhave activity against a target are termed drug leads or candidates.

The true functional targets for therapeutic interventions are notproteins or genes in isolation, however, but are molecular flux ratesthrough fully assembled pathways in the intact biochemical pathway ornetwork. In the final analysis, it is only the flow of molecules througha pathway in the living system, rather than the activity of anycatalytic protein or encoding gene in isolation, that has functionalconsequences for a disease or, indeed, for any organismal phenotype.Moreover, the key regulated parameter in all biochemical systems is fluxrates, whether one is discussing enzymatic catalysis or control ofcomplex pathways (see Kacser, H. & Burns, J. A. (1973). The control offlux. In Rate Control of Biological Processes. Symposium of the Societyfor Experimental Biology Vol 27 (ed. D. D. Davies), pp. 65-104.Cambridge University Press, Cambridge). The kinetic biomarkers formeasuring molecular flux rates through biochemical pathways and networksin vivo in fully assembled living systems thereby differ from currentlyavailable non-kinetic, measurement tools used in DDA and in medicaldiagnosis.

A new measurable target or biomarker of compound action that has broadapplications in pharmaceutical research and development, in clinicalmedicine, and public health is disclosed herein. In the case of DDA,instead of measuring the concentration, structure, state, activity, orcomposition of proteins, genes, metabolites or other components ofbiological systems as targets of drug action, as is currently taught forDDA and medical diagnosis, the Applicant has discovered that molecularflux rates through targeted critical metabolic pathways in vivo,measured by introduction of isotope labels, such as stable isotopelabels, into a living system, are informative, higher-level targets ofdrug action and functional measures of disease activity with powerfulutility in DDA and clinical medicine.

The methods described herein involve the following steps: identifying abiochemical pathway flux rate that is a potentially critical target foraction of a drug in a disease or condition of interest; identifying atarget molecule in the metabolic pathway of interest whose kinetics(synthesis, breakdown, input, removal, or turnover) can be used torepresent molecular flux rates through the critical metabolic pathway ofinterest; introducing an isotopic label, such as a non-radioactive(stable) isotope label, into the living system of interest that isdesigned to result in the formation in vivo of an isotopically perturbedpopulation of the target molecule not otherwise present in nature;isolating the target molecule of interest and measuring its isotopiccontent and/or pattern, or rate of change of isotopic content and/orpattern, for example by use of mass spectrometry; determining thekinetics of the target molecule of interest, based on its perturbedisotopic content and/or pattern or rate of change of isotopic contentand/or pattern and, thereby, calculating molecular flux rates throughthe targeted metabolic pathway of interest; testing and determining theeffect of a compound (e.g., chemical entity (new or old), a drugcandidate, a drug lead, an already-approved drug, a biological factor),or combinations or mixtures thereof, on molecular flux rates through themetabolic pathway of interest in model systems or humans with or withoutthe disease of interest. In this manner, the pathway flux ratemeasurement can be used as a biomarker of compound action in the diseaseof interest, and the activity of compounds or combinations thereof onmolecular flux rates through the targeted metabolic pathway in theliving system of interest can be used for identifying potentialtherapeutic or toxic actions of a compound or combinations thereof.

Procedures for validating the use of molecular flux rates through apathway (measured by the stable isotope labeling/isotopic measurementmethod disclosed herein) as a target of compound action for specificdiseases of interest, are also disclosed. Also disclosed is thecapability of applying the same or closely related stable isotopelabeling procedures described herein at all levels of the DDA chain,from cells to human subjects, and from pre-clinical studies to phase IVclinical trials and subsequent routine medical care. The applicationdescribed herein provides a large number of advantages over currentlyavailable non-kinetic biochemical measurement tools for DDA and medicalcare.

In another embodiment, the application includes the following steps:identifying a biochemical pathway flux rate that is a potentiallycritical target for action of a compound in a disease or condition ofpublic health interest; identifying a target molecule in the metabolicpathway whose kinetics (synthesis, breakdown, input, removal, orturnover) can be used to represent molecular flux rates through themetabolic pathway; introducing an isotopic label, such as anon-radioactive (stable) isotope label, into the living system ofinterest that is designed to result in the formation in vivo of anisotopically perturbed population of the target molecule not otherwisepresent in nature; isolating the target molecule of interest andmeasuring its isotopic content and/or pattern, or rate of change ofisotopic content and/or pattern, for example by use of massspectrometry; determining the kinetics of the target molecule ofinterest, based on its perturbed isotopic content and/or pattern or rateof change of isotopic content and/or pattern and, thereby, calculatingmolecular flux rates through the targeted metabolic pathway; testing anddetermining the effect of a compound such as a chemical entity (new orold), an industrial chemical, food additive, environmental pollutant,cosmetic, biological factor, or combinations or mixtures thereof, onmolecular flux rates through the metabolic pathway in model systems suchas cultured cell systems and animals. In this manner, the pathway fluxrate measurement can be used as a biomarker of chemical or biologicalaction in the public health disease of interest, and the activity ofindustrial chemicals, food additives, cosmetics, environmentalpollutants, or biological factors, or combinations thereof on molecularflux rates through the targeted metabolic pathway in the living systemof interest can be used for identifying potential toxic actions of theindustrial chemicals, cosmetics, food additives, environmentalpollutants, biological factors, or combinations thereof.

Disclosed herein are methods for testing the effects of compounds suchas chemical entities (new or old), drug candidates, drug leads,already-approved drugs, biological factors, or combinations or mixturesthereof on molecular flux rates through metabolic pathways in livingsystems as biomarkers for DDA and medical diagnosis. Also disclosed aremethods for testing the effects of compounds such as chemical entities(new or old), industrial chemicals, food additives, cosmetics, andenvironmental pollutants on molecular flux rates through metabolicpathways in living systems as biomarkers of chemically-induced orbiologically-induced disease or injury (i.e., occupational or industrialtoxicological, food toxicological, dermatotoxicological, andenvironmental toxicological applications).

II. General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press,Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); and Mass isotopomer distribution analysis ateight years: theoretical, analytic and experimental considerations byHellerstein and Neese (Am J Physiol 276 (Endocrinol Metab. 39)E1146-E1162, 1999). Furthermore, procedures employing commerciallyavailable assay kits and reagents will typically be used according tomanufacturer-defined protocols unless otherwise noted.

III. Definitions

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, Massisotopomer distribution analysis at eight years: theoretical, analyticand experimental considerations by Hellerstein and Neese (Am J Physiol276 (Endocrinol Metab. 39) E1146-E1162, 1999). As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

“Molecular flux rates” refers to the dynamic flow or rate of synthesisand/or breakdown of molecules within a cell, tissue, or organism.“Molecular flux rates” also refers to a molecule's input into or removalfrom a pool of molecules, and is therefore synonymous with the flow intoand out of said pool of molecules.

“Metabolic pathway” refers to any linked series of two or morebiochemical steps in a living system (i.e., a biochemical process), thenet result of which is a chemical, spatial or physical transformation ofa molecule or molecules. Metabolic pathways are defined by the directionand flow of molecules through the biochemical steps that comprise thepathway. Molecules within metabolic pathways can be of any biochemicalclass, e.g., including but not limited to lipids, proteins, amino acids,carbohydrates, nucleic acids, polynucleotides, porphyrins,glycosaminoglycans, glycolipids, intermediary metabolites, inorganicminerals, ions, etc.

“Flux rate through a metabolic pathway” refers to the rate of moleculartransformations through a defined metabolic pathway. The unit of fluxrates through pathways is chemical mass per time (e.g., moles perminute, grams per hour). Flux rate through a pathway optimally refers tothe transformation rate from a clearly defined biochemical startingpoint to a clearly defined biochemical end-point, including all thestages in between in the defined metabolic pathway of interest.

“Isotopes” refer to atoms with the same number of protons and hence ofthe same element but with different numbers of neutrons (e.g., ¹H vs. ²Hor D).

“Isotopologues” refer to isotopic homologues or molecular species thathave identical elemental and chemical compositions but differ inisotopic content (e.g., CH₃NH₂ vs. CH₃NHD in the example above).Isotopologues are defined by their isotopic composition, therefore eachisotopologue has a unique exact mass but may not have a uniquestructure. An isotopologue is usually comprised of a family of isotopicisomers (isotopomers) which differ by the location of the isotopes onthe molecule (e.g., CH₃NHD and CH₂DNH₂ are the same isotopologue but aredifferent isotopomers).

“Isotope-labeled water” includes water labeled with one or more specificheavy isotopes of either hydrogen or oxygen. Specific examples ofisotope-labeled water include ²H₂O, ³H₂O, and H₂ ¹⁸O.

“Chemical entity” includes any chemical, whether new or known, that isadministered to a living system for the purpose of screening it forbiological or biochemical activity toward the goal of discoveringpotential therapeutic agents (drugs or drug candidates or drug leads) oruncovering toxic effects (industrial chemicals, pesticides, herbicides,food additives, cosmetics, and the like).

“Drug leads” or “drug candidates” are herein defined as chemicalentities or biological molecules that are being evaluated as potentialtherapeutic agents (drugs). “Drug agents” or “agents” are usedinterchangeably herein and describe any composition of matter (e.g.,chemical entity or biological factor) that is administered, approved orunder testing as potential therapeutic agent or is a known therapeuticagent.

“Known drugs” or “known drug agents” or “already-approved drugs” refersto compounds (i.e., chemical entities or biological factors) that havebeen approved for therapeutic use as drugs in human beings or animals inthe United States or other jurisdictions. In the context of the presentinvention, the term “already-approved drug” means a drug having approvalfor an indication distinct from an indication being tested for by use ofthe methods disclosed herein. Using psoriasis and fluoxetine as anexample, the methods of the present invention allow one to testfluoxetine, a drug approved by the FDA (and other jurisdictions) for thetreatment of depression, for effects on biomarkers of psoriasis (e.g.,keratinocyte proliferation or keratin synthesis); treating psoriasiswith fluoxetine is an indication not approved by FDA or otherjurisdictions. In this manner, one can find new uses (in this example,anti-psoriatic effects) for an already-approved drug (in this example,fluoxetine).

“Biological factor” refers to a compound or compounds made by livingorganisms having biological or physiological activities (e.g.,preventive, therapeutic and/or toxic effects). Examples of biologicalfactors include, but are not limited to, vaccines, polyclonal ormonoclonal antibodies, recombinant proteins, isolated proteins, solublereceptors, gene therapy products, environmental toxins, and the like. Asused herein, the term “biologics” is synonymous with “biologicalfactor.”

“Compound” means, in the context of the present application, any newchemical entity, chemical entity, drug lead, drug candidate, drug, drugagent, agent, known drug, known drug agent, already-approved drug,biologic, or biological factor, food additives, industrial chemicals,environmental pollutants and the like. The term is meant to encompassall chemical and biological molecules.

“Food additive” includes, but is not limited to, organoleptic agents(i.e., those agents conferring flavor, texture, aroma, and color),preservatives such as nitrosamines, nitrosamides, N-nitroso substancesand the like, congealants, emulsifiers, dispersants, fumigants,humectants, oxidizing and reducing agents, propellants, sequestrants,solvents, surface-acting agents, surface-finishing agents, synergists,pesticides, chlorinated organic compounds, any chemical ingested by afood animal or taken up by a food plant, and any chemical leaching into(or otherwise finding its way into) food or drink from packagingmaterial. The term is meant to encompass those chemicals which are addedinto food or drink products at some step in the manufacturing andpackaging process, or find their way into food by ingestion by foodanimals or uptake by food plants, or through microbial byproducts suchas endotoxins and exotoxins (pre-formed toxins such as botulinin toxinor aflatoxin), or through the cooking process (such as heterocyclicamines, e.g., 2-amino-3-methyllimidazo[4,5-f]quinolone), or by leachingor some other process from packaging material during manufacturing,packaging, storage, and handling activities.

“Industrial chemical” includes, but is not limited to, volatile organiccompounds, semi-volatile organic compounds, cleaners, solvents,thinners, mixers, metallic compounds, metals, organometals, metalloids,substituted and non-substituted aliphatic and acyclic hydrocarbons suchas hexane, substituted and non-substituted aromatic hydrocarbons such asbenzene and styrene, halogenated hydrocarbons such as vinyl chloride,aminoderivatives and nitroderivatives such as nitrobenzene, glycols andderivatives such as propylene glycol, ketones such as cyclohexanone,aldehydes such as furfural, amides and anhydrides such as acrylamide,phenols, cyanides and nitriles, isocyanates, and pesticides, herbicides,rodenticides, and fungicides.

“Environmental pollutant” includes any chemical not found in nature orchemicals that are found in nature but artificially concentrated tolevels exceeding those found in nature (at least found in accessiblemedia in nature). So, for example, environmental pollutants can includeany of the non-natural chemicals identified as an occupational orindustrial chemical yet found in a non-occupational or industrialsetting such as a park, school, or playground. Alternatively,environmental pollutants may comprise naturally occurring chemicals suchas lead but at levels exceeding background (for example, lead found inthe soil along highways deposited by the exhaust from the burning ofleaded gasoline in automobiles). Environmental pollutants may be from apoint source such as a factory smokestack or industrial liquid dischargeinto surface or groundwater, or from a non-point source such as theexhaust from cars traveling along a highway, the diesel exhaust (and allthat it contains) from buses traveling along city streets, or pesticidesdeposited in soil from airborne dust originating in farmlands. As usedherein, “environmental contaminant” is synonymous with “environmentalpollutant.”

“Living system” includes, but is not limited to, cells, cell lines,animal models of disease, guinea pigs, rabbits, dogs, cats, other petanimals, mice, rats, non-human primates, and humans.

A “biological sample” encompasses any sample obtained from a cell,tissue, or organism. The definition encompasses blood and other liquidsamples of biological origin, that are accessible from an organismthrough sampling by invasive means (e.g., surgery, open biopsy,endoscopic biopsy, and other procedures involving non-negligible risk)or by minimally invasive or non-invasive approaches (e.g., urinecollection, blood drawing, needle aspiration, and other proceduresinvolving minimal risk, discomfort or effort). The definition alsoincludes samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components, such as proteins or organicmetabolites. The term “biological sample” also encompasses a clinicalsample such as serum, plasma, other biological fluid, or tissue samples,and also includes cells in culture, cell supernatants and cell lysates.

“Biological fluid” refers, but is not limited to, urine, blood,interstitial fluid, edema fluid, saliva, lacrimal fluid, inflammatoryexudates, synovial fluid, abscess, empyema or other infected fluid,cerebrospinal fluid, sweat, pulmonary secretions (sputum), seminalfluid, feces, bile, intestinal secretions, or other biological fluid.

“Exact mass” refers to mass calculated by summing the exact masses ofall the isotopes in the formula of a molecule (e.g., 32.04847 forCH₃NHD).

“Nominal mass” refers to the integer mass obtained by rounding the exactmass of a molecule.

“Mass isotopomer” refers to family of isotopic isomers that is groupedon the basis of nominal mass rather than isotopic composition. A massisotopomer may comprise molecules of different isotopic compositions,unlike an isotopologue (e.g., CH₃NHD, ¹³CH₃NH₂, CH₃ ¹⁵NH₂ are part ofthe same mass isotopomer but are different isotopologues). Inoperational terms, a mass isotopomer is a family of isotopologues thatare not resolved by a mass spectrometer. For quadrupole massspectrometers, this typically means that mass isotopomers are familiesof isotopologues that share a nominal mass. Thus, the isotopologuesCH₃NH₂ and CH₃NHD differ in nominal mass and are distinguished as beingdifferent mass isotopomers, but the isotopologues CH₃NHD, CH₂DNH₂,¹³CH₃NH₂, and CH₃ ¹⁵NH₂ are all of the same nominal mass and hence arethe same mass isotopomers. Each mass isotopomer is therefore typicallycomposed of more than one isotopologue and has more than one exact mass.The distinction between isotopologues and mass isotopomers is useful inpractice because all individual isotopologues are not resolved usingquadrupole mass spectrometers and may not be resolved even using massspectrometers that produce higher mass resolution, so that calculationsfrom mass spectrometric data must be performed on the abundances of massisotopomers rather than isotopologues. The mass isotopomer lowest inmass is represented as M₀; for most organic molecules, this is thespecies containing all ¹²C, ¹H, ¹⁶O, ¹⁴N, etc. Other mass isotopomersare distinguished by their mass differences from M₀ (M₁, M₂, etc.). Fora given mass isotopomer, the location or position of isotopes within themolecule is not specified and may vary (i.e., “positional isotopomers”are not distinguished).

“Mass isotopomer envelope” refers to the set of mass isotopomerscomprising the family associated with each molecule or ion fragmentmonitored.

“Mass isotopomer pattern” refers to a histogram of the abundances of themass isotopomers of a molecule. Traditionally, the pattern is presentedas percent relative abundances where all of the abundances arenormalized to that of the most abundant mass isotopomer; the mostabundant isotopomer is said to be 100%. The preferred form forapplications involving probability analysis, such as mass isotopomerdistribution analysis (MIDA), however, is proportion or fractionalabundance, where the fraction that each species contributes to the totalabundance is used. The term “isotope pattern” may be used synonymouslywith the term “mass isotopomer pattern.”

“Monoisotopic mass” refers to the exact mass of the molecular speciesthat contains all ¹H, ¹²C, ¹⁴N, ¹⁶O, ³²S, etc. For isotopologuescomposed of C, H, N, O, P, S, F, Cl, Br, and I, the isotopic compositionof the isotopologue with the lowest mass is unique and unambiguousbecause the most abundant isotopes of these elements are also the lowestin mass. The monoisotopic mass is abbreviated as m₀ and the masses ofother mass isotopomers are identified by their mass differences from m₀(m₁, m₂, etc.).

By “derivatize”, “derivatizing”, “derivatization”, “hydrolysis andderivatization”, in the context of the current invention, is meant theprocess of preparing samples for GC/MS analysis. This preparation can beperformed on isolated biomolecules, cells, complex samples, or othersamples or molecules and the specific process varies depending on thepathway being analyzed. Such preparation involves multiple procedures,each with many steps, and usually ends with a “derivatization”procedure. As such, the extended process of sample preparation mayoccasionally be referred to by these terms, as it is the finalprocedure. In context, the term may also refer only to this finalprocedure.

“Isotopically perturbed” refers to the state of an element or moleculethat results from the explicit incorporation of an element or moleculewith a distribution of isotopes that differs from the distribution thatis most commonly found in nature, whether a naturally less abundantisotope is present in excess (enriched) or in deficit (depleted).

By “molecule of interest” is meant any molecule (polymer and/ormonomer), including but not limited to, amino acids, carbohydrates,fatty acids, peptides, sugars, lipids, nucleic acids, polynucleotides,glycosaminoglycans, polypeptides, or proteins that are present within ametabolic pathway within a living system. In the context of the presentinvention, a “molecule of interest” may be a “biomarker” of disease andits flux rate, relative to the flux rate of an unexposed or otherwisehealthy subject (i.e., control subject), may represent clinicallynon-observant or subtle pathophysiological occurrences in a subject ofinterest that may be predictive of future disease or injury in thesubject of interest. In this manner, comparing the flux rates of one ormore biomarkers of interest in a subject of interest with the flux ratesof one or more biomarkers of interest in a control subject, will finduse in diagnosing the subject of interest with, or evaluating orquantifying the subject of interest's risk in acquiring, a disease ofinterest. Moreover, such information will find use in establishing aprognosis for a subject of interest having a disease of interest,monitoring the progression of a disease of interest in a subject ofinterest, or evaluating the therapeutic efficacy of a treatment regimenin a subject of interest having a disease of interest.

By “subject of interest” is meant a human or animal having a disease ofinterest or having some level of risk in acquiring a disease ofinterest.

By “control subject” is meant a human or animal not having the diseaseof interest or not having some level of risk in acquiring the disease ofinterest.

“Monomer” refers to a chemical unit that combines during the synthesisof a polymer and which is present two or more times in the polymer.

“Polymer” refers to a molecule synthesized from and containing two ormore repeats of a monomer. A “biopolymer” is a polymer synthesized by orin a living system or otherwise associated with a living system.

“Protein” refers to a polymer of amino acids. As used herein, a“protein” may refer to long amino acid polymers as well as shortpolymers such as peptides.

By “amino acid” is meant any amphoteric organic acid containing theamino group (i.e., NH₂). The term encompasses the twenty common (oftenreferred in the art as “standard” or sometimes as “naturally occurring”)amino acids as well as the less common (often referred in the art as“nonstandard”) amino acids. Examples of the twenty common amino acidsinclude the alpha-amino acids (or α-amino acids), which have the aminogroup in the alpha position, and generally have the formulaRCH—(NH₂)—COOH. The α-amino acids are the monomeric building blocks ofproteins and can be obtained from proteins through hydrolysis. Examplesof nonstandard amino acids include, but are not limited toγ-aminobutyric acid, dopamine, histamine, thyroxine, citrulline,ornithine, homocysteine, and S-adenosylmethionine.

“Lipid” refers to any of a heterogeneous group of fats and fatlikesubstances characterized by being water insoluble and being extractableby nonpolar (or organic) solvents such as alcohol, ether, chloroform,benzene, etc. All contain as a major constituent aliphatic hydrocarbons.The lipids, which are easily stored in the body, serve as a source offuel, are an important constituent of cell structure, and serve otherbiological functions. Lipids include, but are not limited to fattyacids, neutral fats (e.g., triacylglycerols), waxes and steroids (e.g.,cholesterol). Complex lipids comprise the glycolipids, lipoproteins andphospholipids.

“Fatty acids” are carboxylic acids with long-chain hydrocarbon sidegroups. They are comprised of organic, monobasic acids, which arederived from hydrocarbons by the equivalent of oxidation of a methylgroup to an alcohol, aldehyde, and then acid. Fatty acids can be eithersaturated or unsaturated.

By “DNA” is meant a polymeric form of deoxyribonucleotides (adenine,guanine, thymine, or cytosine) in double-stranded or single-strandedform, either relaxed or supercoiled. This term refers only to theprimary and secondary structure of the molecule, and does not limit itto any particular tertiary forms. Thus, this term includes single- anddouble-stranded DNA found, inter alia, in linear DNA molecules (e.g.,restriction fragments), viruses, plasmids, and chromosomes. The termcaptures molecules that include the four bases adenine, guanine,thymine, or cytosine, as well as molecules that include base analogswhich are known in the art.

A “nucleic acid” sequence refers to a DNA or RNA sequence. The termcaptures sequences that include any of the known base analogues of DNAand RNA such as, but not limited to 4-acetylcytosine,8-hydroxy-N⁶-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N⁶-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N⁶-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

By “carbohydrate” is meant an aldehyde or ketone derivative of astraight-chain polyhydroxyl alcohol containing at least three carbonatoms. The polyhydroxyl alcohol is primarily (but not exclusively) ofthe pentahydric and hexahydric alcohol varieties. Carbohydrates are sonamed because the hydrogen and oxygen are usually in the proportion toform water with the general formula C_(n)(H₂O)_(n). The most importantcarbohydrates are the starches, sugars, celluloses and gums. They areclassified into mono, di, tri, poly and heterosaccharides. The smallestare monosaccharides like glucose whereas polysaccharides such as starch,cellulose or glycogen can be large and indeterminate in length.

By “sugar” is meant the common name for any sweet, crystalline, simplecarbohydrate that is an aldehyde or ketone derivative of a polyhydricalcohol. Sugars are mainly disaccharides like sucrose andmonosaccharides like fructose or glucose. The term encompassesmonosaccharides, disaccharides, trisaccharides, heterosaccharides, orpolysaccharides (which are comprised of monosaccharide residues).Monosaccharides include glucose (both D-glucose and L-glucose), mannose,fructose galactose and sugar derivatives including, but not limited toN-acetylmuramic acid, N-acetylneuraminic acid and other sialic acids,N-acetylmannosamine, glucuronic acid, glucosamine, etc. Polysaccharidesinclude disaccharides such as sucrose, maltose and lactose and longerchain sugar molecules such as starch, glycogen, cellulose, chitin, etc.By the term “oligosaccharide” is meant a molecule comprised of a fewcovalently linked monosaccharide monomers.

By “glycosaminoglycan” is meant a polymer comprised of a network oflong, unbranched chains made up of repeating units of disaccharides thatcontain amino group sugars, at least one of which has a negativelycharged side group (carboxylate or sulfate). Examples ofglycosaminoglycans include, but are not limited to hyaluronate(D-glucuronic acid-N-acetyl-D-glucosamine: MW up to 10 million),chondroitin sulfate (D-glucuronic acid-N-acetyl-D-galactosamine-4 or6-sulfate), dermatan sulfate (D-glucuronic acid or L-iduronicacid-IV-acetyl-D-galactosamine), keratan sulfate(D-galactose-N-acetyl-D-glucosamine sulfate), and heparan sulfate(D-glucuronic acid or L-iduronic acid-N-acetyl-D-glucosamine).“Mucopolysaccharide” is a term that is synonymous withglycosaminoglycan.

By “glycoprotein” is meant a protein or polypeptide that is covalentlylinked to one or more carbohydrate molecules. Glycoproteins includeproteoglycans and many, if not most, of the important integral membraneproteins protruding through the exterior leaflet into the extracellularspace, as well as many, if not most, of the secreted proteins.

By “proteoglycan” is meant any of a diverse group of macromoleculescomprising proteins and glycosaminoglycans. “Mucoprotein” is a term thatis synonymous with proteoglycan.

“Isotope labeled substrate” includes any isotope-labeled precursormolecule that is able to be incorporated into a molecule of interest ina living system. Examples of isotope labeled substrates include, but arenot limited to, ²H₂O, ³H₂O, ²H-glucose, ²H-labeled amino acids,²H-labeled organic molecules, ¹³C-labeled organic molecules, ¹⁴C-labeledorganic molecules, ¹³CO₂, ¹⁴CO₂, ¹⁵N-labeled organic molecules and¹⁵NH₃.

“Labeled sugar” refers to a sugar incorporating a stable isotope labelsuch as one or more ²H isotopes.

“Labeled fatty acid” refers to a fatty acid incorporating a stableisotope label such as one or more ²H isotopes.

“Deuterated water” refers to water incorporating a stable isotope labelsuch as one or more ²H isotopes.

“Labeled glucose” refers to glucose labeled with one or more ²Hisotopes. Specific examples of labeled glucose or ²H-labeled glucoseinclude [6,6-²H₂]glucose, [1-²H₁]glucose, and [1,2,3,4,5,6-²H₇] glucose.

“Exposing” a living system to a compound such as a chemical entity orentities can be from, but is not limited to, topical application, oralingestion, inhalation, subcutaneous injection, intraperitonealinjection, intravenous injection, and intraarterial injection, inanimals or other higher organisms.

By “therapeutic action” is meant an effect on a biochemical or molecularprocess (i.e., the flow of molecules through metabolic pathways ornetworks) that is believed to be responsible for, or contributing in, acausal manner to the initiation, progression, severity, pathology,aggressiveness, grade, activity, disability, mortality, morbidity,disease sub-classification or other underlying pathogenic or pathologicfeature of one or more diseases wherein said effect is beneficial tohealth or otherwise contributes to a desirable outcome (e.g., adesirable clinical outcome).

By “action” is meant a specific and direct consequence of anintervention such as the administering of a drug.

By “effect” is meant any consequence, including secondary or tangential,not only of an intervention with a compound but a consequence of anatural occurrence such as the effect a gene exerts when naturallyexpressed or inhibited.

By “toxic effect” is meant an adverse response by a living systemexposed to a compound or combinations or mixtures thereof. A toxiceffect can include, for example, end-organ toxicity.

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman.

By “mammal” is meant any member of the class Mammalia including, withoutlimitation, humans and nonhuman primates such as chimpanzees and otherapes and monkey species; farm animals such as cattle, sheep, pigs, goatsand horses; domestic mammals such as dogs and cats; laboratory animalsincluding rodents such as mice, rats and guinea pigs, and the like. Theterm does not denote a particular age or sex. Thus, adult and newbornsubjects, as well as fetuses, whether male or female, are intended to becovered.

“At least partially identified” in the context of drug discovery anddevelopment means at least one clinically relevant pharmacologicalcharacteristic of a drug agent (i.e., a “compound”) has been identifiedusing one or more of the methods of the present invention. Thischaracteristic may be a desirable one, for example, increasing ordecreasing molecular flux rates through a metabolic pathway thatcontributes to a disease process, altering signal transduction pathwaysor cell surface receptors that alter the activity of metabolic pathwaysrelevant to a disease, inhibiting activation of an enzyme and the like.Alternatively, a pharmacological characteristic of a drug agent may bean undesirable one for example, the production of one or more toxiceffects. There are a plethora of desirable and undesirablecharacteristics of drug agents well known to those skilled in the artand each will be viewed in the context of the particular drug agentbeing developed and the targeted disease. A drug agent can be more thanat least partially identified when, for example, several characteristicshave been identified (desirable or undesirable or both) that aresufficient to support a particular milestone decision point along thedrug development pathway. Such milestones include, but are not limitedto, pre-clinical decisions for in vitro to in vivo transition, pre-INDfiling go/no go decision, phase I to phase II transition, phase II tophase III transition, NDA filing, and FDA approval for marketing.Therefore, “at least partially” identified includes the identificationof one or more pharmacological characteristics useful in evaluating adrug agent in the drug discovery/drug development process. Apharmacologist or physician or other researcher may evaluate all or aportion of the identified desirable and undesirable characteristics of adrug agent to establish its therapeutic index. This may be accomplishedusing procedures well known in the art.

“Manufacturing a drug agent” in the context of the present inventionincludes any means, well known to those skilled in the art, employed forthe making of a drug agent product. Manufacturing processes include, butare not limited to, medicinal chemical synthesis (i.e., syntheticorganic chemistry), combinatorial chemistry, biotechnology methods suchas hybridoma monoclonal antibody production, recombinant DNA technology,and other techniques well known to the skilled artisan. Such a productmay be a final drug agent that is marketed for therapeutic use, acomponent of a combination product that is marketed for therapeutic use,or any intermediate product used in the development of the final drugagent product, whether as part of a combination product or a singleproduct. “Manufacturing drug agent” is synonymous with “manufacturing acompound.”

By “authentic biomarker” is meant a physical, biochemical, orphysiologic measurement from or on the organism that represents a trueor intended mechanistic target of a compound or a mechanistic eventbelieved to be responsible for, or contributing in, a causal manner tothe initiation, progression, severity, pathology, aggressiveness, grade,activity, disability, mortality, morbidity, disease sub-classificationor other underlying pathogenic or pathologic feature of one or morediseases. A biomarker may be the target for monitoring the outcome of atherapeutic intervention (i.e., the functional or structural target of adrug agent). As defined herein “authentic biomarker” and “biomarkers”are used interchangeably herein and refer to biochemical processes thatare involved in, or are believed to be involved in, the etiology orprogression of a disease or disorder. The biochemical process (i.e., theflow of molecules through a targeted metabolic pathway or network) isthe focus of analysis (as disclosed herein) since it is the underlyingchanges of the biochemical process (i.e., molecular flux rates) that maybe the significant or authentic target for treatment or diagnosticmonitoring of the disease or disorder.

By “surrogate biomarker” is meant a physical, biochemical, orphysiologic measurement from or on the organism that is often acceptedby governmental agencies (e.g., FDA) or medical opinion to be asufficient therapeutic target in its own right, independent of “hard”clinical outcomes such as mortality, lost work days, morbidity, etc.There are relatively few accepted surrogate biomarkers in the U.S. andthese include blood pressure and blood glucose levels. Such surrogatebiomarkers are not the subject of this application.

By “evaluate” or “evaluation” or “evaluating,” in the context of thepresent invention, is meant a process whereby the activity, toxicity,relative potency, potential therapeutic value and/or efficacy,significance, or worth of a chemical entity, biological factor,combination of chemical entities, or combination of biological factorsis determined through appraisal and study, usually by means of comparingexperimental outcomes to established standards and/or conditions. Theterm embraces the concept of providing sufficient information for adecision-maker to make a “go/no go” decision on a chemical entity orbiological factor (or combinations of chemical entities or combinationsof biological factors) to proceed further in the drug developmentprocess. A “go/no go” decision may be made at any point or milestone inthe drug development process including, but not limited to, any stagewithin pre-clinical development, the pre-clinical to Investigational NewDrug (IND) stage, the Phase I to Phase II stage, the Phase II to moreadvanced phases within Phase II (such as Phase IIb), the Phase H toPhase III stage, the Phase III to the New Drug Application (NDA) orBiologics License Application (BLA) stage, or stages beyond (such asPhase IV or other post-NDA or post-BLA stages). The term also embracesthe concept of providing sufficient information to select“best-in-breed” (or “best-of-breed”) in a class of compounds (chemicalentities, biologics).

By “characterize,” “characterizing,” or “characterization,” in thecontext of the present invention is meant an effort to describe thecharacter or quality of a chemical entity or combination of chemicalentities. As used herein, the term is nearly equivalent to “evaluate,”yet lacks the more refined aspects of “evaluate,” in which to “evaluate”a drug includes the ability to make a “go/no go” decision (based on anassessment of therapeutic value) on proceeding with that drug orchemical entity through the drug development process.

By “hepatic fibrosis” is meant any fibrotic condition of the liver thatimpairs hepatocyte or hepatic function and thus total liver function.The fibrosis need not have as its origin the hepatic tissue but mayarise, for example, around hepatic venules, which results in adisturbance of blood flow to the hepatocytes. Hepatic fibrosis iscommonly termed “fibrosis;” “fibrosis,” “liver fibrosis,” and “hepaticfibrosis,” as used herein, are equivalent terms.

By “condition” or “medical condition” is meant the physical status ofthe body as a whole or of one of its parts. The term is usually used toindicate a change from a previous physical or mental status, or anabnormality not recognized by medical authorities as a disease ordisorder. Examples of “conditions” or “medical conditions” includeobesity and pregnancy.

IV. Methods of the Invention A. Overview of the Methods of the Invention

The present invention is directed to methods of measuring changes in themolecular flux rates of one or more molecules in one or more metabolicpathways of interest within a living system. The metabolic pathways ofinterest are either known or suspected to be important as drivingfactors for, or fundamental mechanisms of, diseases or disorders.Changes in metabolic pathways of interest (i.e., molecular flux rates,kinetics) can be elicited by one or more compounds including chemicalentities for example, known drugs, drug candidates, drug leads (orcombinations thereof), or industrial chemicals such as pesticides,herbicides, plastics, and the like, or cosmetics, or food additives.

At least one isotope-labeled substrate molecule is administered to acell, tissue or organism for a period of time sufficient to beincorporated in vivo (or intracellularly if the living system is acultured cell) into one or more molecules of interest within one or moretargeted metabolic pathways. In one embodiment, the isotope-labeledsubstrate molecules are labeled with a stable isotope (i.e.,non-radioactive isotope). In another embodiment, the isotope-labeledsubstrate molecule is labeled with a radioactive isotope. In yet anotherembodiment, both stable and radioactive isotopes are used to label oneor more isotope-labeled substrate molecules.

The targeted molecule of interest is obtained by biochemical isolationprocedures from the cell, tissue, or organism, and is identified by massspectrometry or by other means known in the art. For methods employingstable isotope labels, the relative and absolute abundances of the ionswithin the mass isotopomeric envelope corresponding to each identifiedmolecule of interest (i.e., the isotopic content and/or pattern of themolecule or the rate of change of the isotopic content and/or pattern ofthe molecule) are quantified. In one embodiment, the relative andabsolute abundances of the ions within the mass isotopomeric envelopecorresponding to each identified molecule of interest are quantified bymass spectrometry. Flux rates through the targeted metabolic pathwaysare then calculated by use of equations known in the art and discussed,infra. Flux rates through the targeted metabolic pathways are comparedin the presence or absence of exposure to one or more compounds, one ormore chemical entities (i.e., drugs, drug candidates, industrialchemicals, food additives, environmental pollutants, and the like) orcombinations of chemical entities (i.e., combinations of drugs, drugcandidates, or other chemicals), or in response to different levels ofexposure to one or more compounds or one or more chemical entities, orin response to different levels of exposure to combinations of compoundsor chemical entities.

In this manner, changes in the targeted underlying biochemical(metabolic) pathways are measured and quantified and related to diseasediagnosis; disease prognosis; therapeutic efficacy of administeredcompounds, drugs, drug candidates, or drug leads; or toxic effects ofcompounds, chemical entities such as drug candidates, drug leads, knowndrugs, industrial chemicals, pesticides, herbicides, cosmetics, foodadditives, and the like.

B. Administering Isotope-Labeled Precursor(s)

As a first step in the methods of the invention, isotope-labeledprecursors are administered.

1. Administering an Isotope-Labeled Precursor Molecule

Modes of administering the one or more isotope-labeled substrates mayvary, depending upon the absorptive properties of the isotope-labeledsubstrate and the specific biosynthetic pool into which each compound istargeted. Precursors may be administered to organisms, plants andanimals including humans directly for in vivo analysis. In addition,precursors may be administered in vitro to living cells. Specific typesof living cells include hepatocytes, adipocytes, myocytes, fibroblasts,microglia, neurons, neuroprogenitor cells, sperm cells, pancreaticβ-cells, intestinal epithelial cells, breast epithelial cells, prostateepithelial cells, endothelial cells, leukocytes, lymphocytes,erythrocytes, microbial cells and any other cell-type that can bemaintained alive and functional in vitro.

Generally, an appropriate mode of administration is one that produces asteady state level of precursor within the biosynthetic pool and/or in areservoir supplying such a pool for at least a transient period of time.Intravascular or oral routes of administration are commonly used toadminister such precursors to organisms, including humans. Other routesof administration, such as subcutaneous or intra-muscularadministration, optionally when used in conjunction with slow releaseprecursor compositions, are also appropriate. Compositions for injectionare generally prepared in sterile pharmaceutical excipients.

a. Labeled Precursor Molecules

(1) Isotope Labels

The first step in measuring molecular flux rates involves administeringan isotope-labeled precursor molecule to a cell, tissue, or organism.The isotope labeled precursor molecule may contain a stable isotope or aradioisotope. Isotope labels that can be used in accordance with themethods of the present invention include, but are not limited to, ²H,¹³C, ¹⁵N, ¹⁸O, ³H, ¹⁴C, ³⁵S, ³²P, ³³P, ¹²⁵I, ¹³¹I, or other isotopes ofelements present in organic systems. These isotopes, and others, aresuitable for all classes of chemicals (i.e., precursor molecules)envisioned for use in the present invention. Such precursor moleculesinclude, but are not limited to, protein precursors, lipid precursors,carbohydrate precursors, nucleic acid precursors, porphyrin precursors,glycosaminoglycan precursors, and proteoglycan precursors (see examplesof each, infra).

In one embodiment, the isotope label is ²H.

(2) Precursor Molecules (Isotope-Labeled Substrates)

The precursor molecule may be any molecule having an isotope label thatis incorporated into a molecule of interest by passage through ametabolic pathway in vivo in a living system (or in vitro in a culturedcell). Precursor molecules typically used include, without limitation:H₂O; CO₂; NH₃; acetyl CoA (to form cholesterol, fatty acids);ribonucleic acids (to form RNA); deoxyribonucleic acids (to form DNA);glucose (to form glycogen); amino acids (to form peptides/proteins);phosphoenol-pyruvate (to form glucose/UDP-glucose); andglycine/succinate (to form porphyrin derivatives). Isotope labels may beused to modify all precursor molecules disclosed herein to formisotope-labeled precursor molecules.

The entire precursor molecule may be incorporated into one or moremolecules of interest within a metabolic pathway. Alternatively, aportion of the precursor molecule may be incorporated into one or moremolecules of interest.

i. Protein Precursors

A protein precursor molecule may be any protein precursor molecule knownin the art. These precursor molecules may be amino acids, CO₂, NH₃,glucose, lactate, H₂O, acetate, and fatty acids.

The isotope label may include specific heavy isotopes of elementspresent in biomolecules, such as ²H, ¹³C, ¹⁵N, ¹⁸O, ³³S, ³⁴S, or maycontain other isotopes of elements present in biomolecules such as ³H,¹⁴C, ³⁵S, ³²P, ³³P, ¹²⁵I, or ¹³¹I.

Precursor molecules of proteins may also include one or more aminoacids. The precursor may be any amino acid. The precursor molecule maybe a singly or multiply deuterated amino acid. The precursor moleculemay be one or more of ¹³C-lysine, ¹⁵N-histidine, ¹³C-serine,¹³C-glycine, ²H-leucine, ¹⁵N-glycine, ¹³C-leucine, ²H₅-histidine, andany deuterated amino acid. By way of example, isotope labeled proteinprecursors include, but are not limited to ²H₂O, ¹⁵NH₃, ¹³CO₂, H¹³CO₃,²H-labeled amino acids, ¹³C labeled amino acids, ¹⁵N labeled aminoacids, ¹⁸O labeled amino acids, ³³S or ³⁴S labeled amino acids, ³H₂O,³H-labeled amino acids, and ¹⁴C labeled amino acids. Labeled amino acidsmay be administered, for example, undiluted or diluted with non-labeledamino acids. All isotope labeled precursors may be purchasedcommercially, for example, from Cambridge Isotope Labs (Andover, Mass.).

Protein precursor molecules may also include any precursor forpost-translational or pre-translationally modified amino acids. Theseprecursors include but are not limited to precursors of methylation suchas glycine, serine or H₂O; precursors of hydroxylation, such as H₂O orO₂; precursors of phosphorylation, such as phosphate, H₂O or O₂;precursors of prenylation, such as fatty acids, acetate, H₂O, ethanol,ketone bodies, glucose, or fructose; precursors of carboxylation, suchas CO₂, O₂, H₂O, or glucose; precursors of acetylation, such as acetate,ethanol, glucose, fructose, lactate, alanine, H₂O, CO₂, or O₂; and otherpre or post-translational modifications known in the art.

The degree of labeling present in free amino acids may be determinedexperimentally, or may be assumed based on the number of labeling sitesin an amino acid. For example, when using hydrogen isotopes as a label,the labeling present in C—H bonds of free amino acid or, morespecifically, in tRNA-amino acids, during exposure to ²H₂O in body watermay be identified. The total number of C—H bonds in each non essentialamino acid is known—e.g., 4 in alanine, 2 in glycine, etc.

The precursor molecule for proteins may be water. The hydrogen atoms onC—H bonds are the hydrogen atoms on amino acids that are useful formeasuring protein synthesis from ²H₂O since the O—H and N—H bonds ofproteins are labile in aqueous solution. As such, the exchange of²H-label from ²H₂O into O—H or N—H bonds occurs without the synthesis ofproteins from free amino acids as described above. C—H bonds undergoincorporation from H₂O into free amino acids during specificenzyme-catalyzed intermediary metabolic reactions. The presence of²H-label in C—H bonds of protein-bound amino acids after ²H₂Oadministration therefore means that the protein was assembled from aminoacids that were in the free form during the period of ²H₂Oexposure—i.e., that the protein is newly synthesized. Analytically, theamino acid derivative used must contain all the C—H bonds but mustremove all potentially contaminating N—H and O—H bonds.

Hydrogen atoms from body water may be incorporated into free aminoacids. ²H or ³H from labeled water can enter into free amino acids inthe cell through the reactions of intermediary metabolism, but ²H or ³Hcannot enter into amino acids that are present in peptide bonds or thatare bound to transfer RNA. Free essential amino acids may incorporate asingle hydrogen atom from body water into the α-carbon C—H bond, throughrapidly reversible transamination reactions. Free non-essential aminoacids contain a larger number of metabolically exchangeable C—H bonds,of course, and are therefore expected to exhibit higher isotopicenrichment values per molecule from ²H₂O in newly synthesized proteins.

One of skill in the art will recognize that labeled hydrogen atoms frombody water may be incorporated into other amino acids via otherbiochemical pathways. For example, it is known in the art that hydrogenatoms from water may be incorporated into glutamate via synthesis of theprecursor α-ketoglutarate in the citric acid cycle. Glutamate, in turn,is known to be the biochemical precursor for glutamine, proline, andarginine. By way of another example, hydrogen atoms from body water maybe incorporated into post-translationally modified amino acids, such asthe methyl group in 3-methyl-histidine, the hydroxyl group inhydroxyproline or hydroxylysine, and others. Other amino acid synthesispathways are known to those of skill in the art.

Oxygen atoms (H₂ ¹⁸O) may also be incorporated into amino acids throughenzyme-catalyzed reactions. For example, oxygen exchange into thecarboxylic acid moiety of amino acids may occur during enzyme catalyzedreactions. Incorporation of labeled oxygen into amino acids is known toone of skill in the art. Oxygen atoms may also be incorporated intoamino acids from ¹⁸O₂ through enzyme catalyzed reactions (includinghydroxyproline, hydroxylysine or other post-translationally modifiedamino acids).

Hydrogen and oxygen labels from labeled water may also be incorporatedinto amino acids through post-translational modifications. In oneembodiment, the post-translational modification may already includelabeled hydrogen or oxygen through biosynthetic pathways prior topost-translational modification. In another embodiment, thepost-translational modification may incorporate labeled hydrogen,oxygen, carbon, or nitrogen from metabolic derivatives involved in thefree exchange labeled hydrogens from body water, either before or afterpost-translational modification step (e.g., methylation, hydroxylation,phosphorylation, prenylation, sulfation, carboxylation, acetylation orother known post-translational modifications).

Protein precursors that are suitable for administration into a subjectinclude, but are not limited to H₂O, CO₂, NH₃ and HCO₃, in addition tothe standard amino acids found in proteins.

The individual being administered a labeled protein precursor may be amammal. In one variation, the individual may be an experimental animalincluding, without limitation, a rodent, primate, hamster, guinea pig,dog, or pig. In variations involving the administering of drugs, drugcandidates, drug leads, or combinations thereof, the individual may be amammal, such as an experimental animal, including an accepted animalmodel of disease, or a human. In variations involving the administeringof food additives, industrial or occupational chemicals, environmentalpollutants, or cosmetics, the individual may be any experimental animalsuch as, without limitation, a rodent, primate, hamster, guinea pig,dog, or pig.

ii. Precursors of Organic Metabolites

Precursors of organic metabolites may be any precursor molecule capableof entering into the organic metabolite pathway. Organic metabolites andorganic metabolite precursors include, but are not limited to, H₂O, CO₂,NH₃, HCO₃, amino acids, monosaccharides, carbohydrates, lipids, fattyacids, nucleic acids, glycolytic intermediates, acetic acid, andtricarboxylic acid cycle intermediates.

Isotope labeled organic metabolite precursors include, but are notlimited to, ²H₂O, ¹⁵NH₃, ¹³CO₂, H¹³CO₃, ²H-labeled amino acids,¹³C-labeled amino acids, ¹⁵N-labeled amino acids, ¹⁸O-labeled aminoacids, ³³S or ³⁴S-labeled amino acids, ³H₂O, ³H-labeled amino acids,¹⁴C-labeled amino acids, ¹⁴CO₂, and H¹⁴CO₂.

Organic metabolite precursors may also be administered directly. Massisotopes that may be useful in mass isotope labeling of organicmetabolite precursors include, but are not limited to, ²H, ³H, ¹³C, ¹⁴C,¹⁵N, ¹⁸O, ³³S, ³⁴S, ³⁵S, ³²P, ¹²⁵I, ¹³¹I, or other isotopes of elementspresent in organic systems. It is often desirable, in order to avoidmetabolic loss of isotope labels, that the isotope-labeled atom(s) berelatively non-labile or at least behave in a predictable manner withinthe subject. By administering the isotope-labeled precursors to thebiosynthetic pool, the isotope-labeled precursors can become directlyincorporated into organic metabolites formed in the pool.

The individual being administered a labeled organic metabolite precursormay be a mammal. In one variation, the individual may be an experimentalanimal including, without limitation, a rodent, primate, hamster, guineapig, dog, or pig. In variations involving the administering of drugs,drug candidates, drug leads, or combinations thereof, the individual maybe a mammal, such as an experimental animal, including an acceptedanimal model of disease, or a human. In variations involving theadministering of food additives, industrial or occupational chemicals,environmental pollutants, or cosmetics, the individual may be anyexperimental animal such as, without limitation, a rodent, primate,hamster, guinea pig, dog, or pig.

iii. Precursors of Nucleic Acids

Precursors of nucleic acids (i.e., RNA, DNA) are any compounds suitablefor incorporation into RNA and/or DNA synthetic pathways. Examples ofsubstrates useful in labeling the deoxyribose ring of DNA include, butare not limited to, [6,6-²H₂] glucose, [U-¹³C₅] glucose and [2-¹³C₁]glycerol (see U.S. Pat. No. 6,461,806, herein incorporated byreference). Labeling of the deoxyribose is superior to labeling of theinformation-carrying nitrogen bases in DNA because it avoids variabledilution sources. The stable isotope labels are readily detectable bymass spectrometric techniques.

In one embodiment, a stable isotope label is used to label thedeoxyribose ring of DNA from glucose, precursors of glucose-6-phosphateor precursors of ribose-5-phosphate. In embodiments where glucose isused as the starting material, suitable labels include, but are notlimited to, deuterium-labeled glucose such as [6,6-²H₂] glucose, [1-²H₁]glucose, [3-²H₁] glucose, [²H₇] glucose, and the like; ¹³C-1 labeledglucose such as [1-¹³C₁] glucose, [U-¹³C₆] glucose and the like; and¹⁸O-labeled glucose such as [1-¹⁸O₂] glucose and the like.

In embodiments where a glucose-6-phosphate precursor or aribose-5-phosphate precursor is desired, a gluconeogenic precursor or ametabolite capable of being converted to glucose-6-phosphate orribose-5-phosphate can be used. Gluconeogenic precursors include, butare not limited to, ¹³C-labeled glycerol such as [2-¹³C₁] glycerol andthe like, a ¹³C-labeled amino acid, deuterated water (²H₂O) and¹³C-labeled lactate, alanine, pyruvate, propionate or other non-aminoacid precursors for gluconeogenesis. Metabolites which are converted toglucose-6-phosphate or ribose-5-phosphate include, but are not limitedto, labeled (²H or ¹³C) hexoses such as [1-²H₁] galactose, [U-¹³C]fructose and the like; labeled (²H or ¹³C) pentoses such as [1-¹³C₁]ribose, [1-²H₁] xylitol and the like, labeled (²H or ¹³C) pentosephosphate pathway metabolites such as [1-²H₁] seduheptalose and thelike, and labeled (²H or ¹³C) amino sugars such as [U-¹³C] glucosamine,[1-²H₁] N-acetyl-glucosamine and the like.

The present invention also encompasses stable isotope labels which labelpurine and pyrimidine bases of DNA through the de novo nucleotidesynthesis pathway. Various building blocks for endogenous purinesynthesis can be used to label purines and they include, but are notlimited to, ¹⁵N-labeled amino acids such as [¹⁵N] glycine, [¹⁵N]glutamine, [¹⁵N] aspartate and the like, ¹³C-labeled precursors such as[1-¹³C₁] glycone, [3-¹³C₁]acetate, [¹³C]HCO₃, [¹³C] methionine and thelike, and H-labeled precursors such as ²H₂O. Various building blocks forendogenous pyrimidine synthesis can be used to label pyrimidines andthey include, but are not limited to, ¹⁵N-labeled amino acids such as[¹⁵N] glutamine and the like, ¹³C-labeled precursors such as [¹³C]HCO₃,[U-¹³C₄] aspartate and the like, and ²H-labeled precursors (²H₂O).

It is understood by those skilled in the art that in addition to thelist above, other stable isotope labels which are substrates orprecursors for any pathways which result in endogenous labeling of DNAare also encompassed within the scope of the invention. The labelssuitable for use in the present invention are generally commerciallyavailable or can be synthesized by methods well known in the art.

The individual being administered a labeled nucleic acid precursor maybe a mammal. In one variation, the individual may be an experimentalanimal including, without limitation, a rodent, primate, hamster, guineapig, dog, or pig. In variations involving the administering of drugs,drug candidates, drug leads, biologics, or combinations thereof, theindividual may be a mammal, such as an experimental animal, including anaccepted animal model of disease, or a human. In variations involvingthe administering of food additives, industrial or occupationalchemicals, environmental pollutants, or cosmetics, the individual may beany experimental animal such as, without limitation, a rodent, primate,hamster, guinea pig, dog, or pig.

iv. Water as a Precursor Molecule

Water is a precursor of proteins and many organic metabolites. As such,labeled water may serve as a precursor in the methods taught herein.

H₂O availability is probably never limiting for biosynthetic reactionsin a cell (because H₂O represents close to 70% of the content of cells,or >35 Molar concentration), but hydrogen and oxygen atoms from H₂Ocontribute stoichiometrically to many reactions involved in biosyntheticpathways:

e.g.: R—CO—CH₂—COOH+NADPH+H₂O→R—CH₂CH₂COOH (fatty acid synthesis).

As a consequence, isotope labels provided in the form of H- orO-isotope-labeled water is incorporated into biological molecules aspart of synthetic pathways. Hydrogen incorporation can occur in twoways: into labile positions in a molecule (i.e., rapidly exchangeable,not requiring enzyme catalyzed reactions) or into stable positions(i.e., not rapidly exchangeable, requiring enzyme catalysis). Oxygenincorporation occurs in stable positions.

Some of the hydrogen-incorporating steps from cellular water into C—Hbonds in biological molecules only occur during well-definedenzyme-catalyzed steps in the biosynthetic reaction sequence, and arenot labile (exchangeable with solvent water in the tissue) once presentin the mature end-product molecules. For example, the C—H bonds onglucose are not exchangeable in solution. In contrast, each of thefollowing C—H positions exchanges with body water during reversal ofspecific enzymatic reactions: C-1 and C-6, in the oxaloacetate/succinatesequence in the Krebs' cycle and in the lactate/pyruvate reaction; C-2,in the glucose-6-phosphate/fructose-6-phosphate reaction; C-3 and C-4,in the glyceraldehyde-3-phosphate/dihydroxyacetone-phosphate reaction;C-5, in the 3-phosphoglycerate/glyceraldehyde-3-phosphate andglucose-6-phosphate/fructose-6-phosphate reactions.

Labeled hydrogen or oxygen atoms from water that are covalentlyincorporated into specific non-labile positions of a molecule therebyreveals the molecule's “biosynthetic history”—i.e., label incorporationsignifies that the molecule was synthesized during the period thatisotope-labeled water was present in cellular water.

The labile hydrogens (non-covalently associated or present inexchangeable covalent bonds) in these biological molecules do not revealthe molecule's biosynthetic history. Labile hydrogen atoms can be easilyremoved by incubation with unlabelled water (H₂O) (i.e., by reversal ofthe same non-enzymatic exchange reactions through which ²H or ³H wasincorporated in the first place), however:

As a consequence, potentially contaminating hydrogen label that does notreflect biosynthetic history, but is incorporated via non-syntheticexchange reactions, can easily be removed in practice by incubation withnatural abundance H₂O.

Analytic methods are available for measuring quantitatively theincorporation of labeled hydrogen atoms into biological molecules (e.g.,liquid scintillation counting for ³H; mass spectrometry or NMRspectroscopy for ²H and ¹⁸O). For further discussions on the theory ofisotope-labeled water incorporation, see, for example, Jungas R L.Biochemistry. 1968 7:3708-17, incorporated herein by reference.

Labeled water may be readily obtained commercially. For example, ²H₂Omay be purchased from Cambridge Isotope Labs (Andover, Mass.), and ³H₂Omay be purchased, e.g., from New England Nuclear, Inc. In general, ²H₂Ois non-radioactive and thus, presents fewer toxicity concerns thanradioactive ³H₂O. ²H₂O may be administered, for example, as a percent oftotal body water, e.g., 1% of total body water consumed (e.g., for 3liters water consumed per day, 30 microliters ²H₂O is consumed). If ³H₂Ois utilized, then a non-toxic amount, which is readily determined bythose of skill in the art, is administered.

Relatively high body water enrichments of ²H₂O (e.g., 1-10% of the totalbody water is labeled) may be achieved relatively inexpensively usingthe techniques of the invention. This water enrichment is relativelyconstant and stable as these levels are maintained for weeks or monthsin humans and in experimental animals without any evidence of toxicity.This finding in a large number of human subjects (>100 people) iscontrary to previous concerns about vestibular toxicities at high dosesof ²H₂O. The Applicant has discovered that as long as rapid changes inbody water enrichment are prevented (e.g., by initial administration insmall, divided doses), high body water enrichments of ²H₂O can bemaintained with no toxicities. For example, the low expense ofcommercially available ²H₂O allows long-term maintenance of enrichmentsin the 1-5% range at relatively low expense (e.g., calculations reveal alower cost for 2 months labeling at 2% ²H₂O enrichment, and thus 7-8%enrichment in the alanine precursor pool, than for 12 hours labeling of²H-leucine at 10% free leucine enrichment, and thus 7-8% enrichment inleucine precursor pool for that period).

Relatively high and relatively constant body water enrichments foradministration of H₂ ¹⁸O may also be accomplished, since the ¹⁸O isotopeis not toxic, and does not present a significant health risk as aresult.

Isotope-labeled water may be administered via continuous isotope-labeledwater administration, discontinuous isotope-labeled wateradministration, or after single or multiple administration ofisotope-labeled water administration. In continuous isotope-labeledwater administration, isotope-labeled water is administered to anindividual for a period of time sufficient to maintain relativelyconstant water enrichments over time in the individual. For continuousmethods, labeled water is optimally administered for a period ofsufficient duration to achieve a steady state concentration (e.g., 3-8weeks in humans, 1-2 weeks in rodents).

In discontinuous isotope-labeled water administration, an amount ofisotope-labeled water is measured and then administered, one or moretimes, and then the exposure to isotope-labeled water is discontinuedand wash-out of isotope-labeled water from body water pool is allowed tooccur. The time course of delabeling may then be monitored. Water isoptimally administered for a period of sufficient duration to achievedetectable levels in biological molecules.

Isotope-labeled water may be administered to an individual or tissue invarious ways known in the art. For example, isotope-labeled water may beadministered orally, parenterally, subcutaneously, intravascularly(e.g., intravenously, intraarterially), or intraperitoneally. Severalcommercial sources of ²H₂O and H₂ ¹⁸O are available, including Isotec,Inc. (Miamisburg Ohio, and Cambridge Isotopes, Inc. (Andover, Mass.).The isotopic content of isotope labeled water that is administered canrange from about 0.001% to about 20% and depends upon the analyticsensitivity of the instrument used to measure the isotopic content ofthe biological molecules. In one embodiment, 4% ²H₂O in drinking wateris orally administered. In another embodiment, a human is administered50 mL of ²H₂O orally.

The individual being administered labeled water may be a mammal. In onevariation, the individual may be an experimental animal including,without limitation, a rodent, primate, hamster, guinea pig, dog, or pig.In variations involving the administering of drugs, drug candidates,drug leads, or combinations thereof, the individual may be a mammal,such as an experimental animal, including an accepted animal model ofdisease, or a human. In variations involving the administering of foodadditives, industrial or occupational chemicals, environmentalpollutants, or cosmetics, the individual may be any experimental animalsuch as, without limitation, a rodent, primate, hamster, guinea pig,dog, or pig.

v. Precursors of Carbohydrates

Compositions comprising carbohydrates may include monosaccharides,polysaccharides, or other compounds attached to monosaccharides orpolysaccharides.

Isotope labels may be incorporated into carbohydrates or carbohydratederivatives. These include monosaccharides (including, but not limitedto, glucose and galactose), amino sugars (such asN-Acetyl-Galactosamine), polysaccharides (such as glycogen),glycoproteins (such as sialic acid) glycolipids (such asgalactocerebrosides), glycosaminoglycans (such as hyaluronic acid,chondroitin-sulfate, and heparan-sulfate) by biochemical pathways knownin the art.

²H-labeled sugars may be administered to an individual asmonosaccharides or as polymers comprising monosaccharide residues.Labeled monosaccharides may be readily obtained commercially (e.g.,Cambridge Isotopes, Massachusetts).

Relatively low quantities of compounds comprising ²H-labeled sugars needbe administered. Quantities may be on the order of milligrams, 10¹ mg,10² mg, 10³ mg, 10⁴ mg, 10⁵ mg, or 10⁶ mg. ²H-labeled sugar enrichmentmay be maintained for weeks or months in humans and in animals withoutany evidence of toxicity. The lower expense of commercially availablelabeled monosaccharides, and low quantity that need to be administered,allow maintenance of enrichments at low expense.

In one embodiment, the labeled sugar is glucose. Glucose is metabolizedby glycolysis and the citric acid cycle. Glycolysis releases most of theH-atoms from C—H bonds of glucose; oxidation via the citric acid cycleensures that all H-atoms are released to H₂O. The loss of ³H— or²H-label by glucose has been used to assess glycolysis, an intracellularmetabolic pathway for glucose. Some investigators have used release of³H from intravenously administered ³H-glucose into ³H₂O as a measure ofglycolysis. Release of ²H-glucose into ²H₂O has not been usedpreviously, because of the expectation that the body water pool is toolarge relative to ²H administration in labeled glucose to achievemeasurable 2H₂O levels. In a further variation, the labeled glucose maybe [6,6-²H₂]glucose, [1-²H₁]glucose, and [1,2,3,4,5,6-²H₇]glucose.

In another embodiment, labeled sugar comprises fructose or galactose.Fructose enters glycolysis via the fructose 1-phosphate pathway, andsecondarily through phosphorylation to fructose 6-phosphate byhexokinase. Galactose enters glycolysis via the galactose to glucoseinterconversion pathway.

Any other sugar is envisioned for use in the present invention.Contemplated monosaccharides include, but are not limited to, trioses,pentoses, hexose, and higher order monosaccharides. Monosaccharidesfurther include, but are not limited to, aldoses and ketoses.

In another embodiment, polymers comprising polysaccharides may beadministered. In yet another embodiment, labeled polysaccharides may beadministered. In yet another embodiment, labeled sugar monomers may beadministered as a component of sucrose (glucose α-(1,2)-fructose),lactose (galactose β-(1,4)-glucose), maltose (glucose α-(1,4)-glucose),starch (glucose polymer), or other polymers.

In one embodiment, the labeled sugar may be administered orally, bygavage, intraperitoneally, intravascularly (e.g., intravenously,intraarterially), subcutaneously, or other bodily routes. In particular,the sugars may be administered to an individual orally, optionally aspart of a food or drink.

The individual being administered a labeled carbohydrate precursor maybe a mammal. In one variation, the individual may be an experimentalanimal including, without limitation, a rodent, primate, hamster, guineapig, dog, or pig. In variations involving the administering of drugs,drug candidates, drug leads, or combinations thereof, the individual maybe a mammal, such as an experimental animal, including an acceptedanimal model of disease, or a human. In variations involving theadministering of food additives, industrial or occupational chemicals,environmental pollutants, or cosmetics, the individual may be anyexperimental animal such as, without limitation, a rodent, primate,hamster, guinea pig, dog, or pig.

vi. Precursors of Lipids and Other Fats

Measuring the metabolism of compounds comprising ²H-labeled fatty acidsare also contemplated by the present invention. Isotope labels fromisotope-labeled water may also be incorporated into fatty acids, theglycerol moiety of acyl-glycerols (including but not limited to,triacylglycerides, phospholipids, and cardiolipin), cholesterol and itsderivatives (including but not limited to cholesterol-esters, bileacids, steroid hormones) by biochemical pathways known in the art.

Complex lipids, such as glycolipids and cerebrosides, can also belabeled from isotope-labeled water, which is a precursor for thesugar-moiety of cerebrosides (including, but not limited to,N-acetylgalactosamine, N-acetylglucosamine-sulfate, glucuronic acid, andglucuronic acid-sulfate).

²H-labeled fatty acids may be administered to an individual as fats orother compounds containing the labeled fatty acids. ²H-labeled fattyacids may be readily obtained commercially. Relatively low quantities oflabeled fatty acids need be administered. Quantities may be on the orderof milligrams, 10¹ mg, 10² mg, 10³ mg, 10⁴ mg, 10⁵ mg, or 10⁶ mg. Fattyacid enrichment, particularly with ²H, may be maintained for weeks ormonths in humans and in animals without any evidence of toxicity. Thelower expense of commercially available labeled fatty acids, and lowquantity that need to be administered, allow maintenance of enrichmentsat low expense.

The release of labeled fatty acids, particularly ²H-fatty acid, tolabeled water, particularly ²H₂O, accurately reflects fat oxidation.Administration of modest amounts of labeled-fatty acid is sufficient tomeasure release of labeled hydrogen or oxygen to water. In particular,administration of modest amounts of ²H-fatty acid is sufficient tomeasure release of ²H to deuterated water.

In another variation, the labeled fatty acids may be administeredorally, by gavage, intraperitoneally, intravascularly (e.g.,intravenously, intraarterially), subcutaneously, or other bodily routes.In particular, the labeled fatty acids may be administered to anindividual orally, optionally as part of a food or drink.

The individual being administered labeled lipid precursors may be amammal. In one variation, the individual may be an experimental animalincluding, without limitation, a rodent, primate, hamster, guinea pig,dog, or pig. In variations involving the administering of drugs, drugcandidates, drug leads, or combinations thereof, the individual may be amammal, such as an experimental animal, including an accepted animalmodel of disease, or a human. In variations involving the administeringof food additives, industrial or occupational chemicals, environmentalpollutants, or cosmetics, the individual may be any experimental animalsuch as, without limitation, a rodent, primate, hamster, guinea pig,dog, or pig.

C. Obtaining One or More Targeted Molecules of Interest

In practicing the methods of the invention, in one aspect, targetedmolecules of interest are obtained from a cell, tissue, or organismaccording to methods known in the art. The methods may be specific tothe particular molecule of interest. Molecules of interest may beisolated from a biological sample.

A plurality of molecules of interest may be acquired from the cell,tissue, or organism. The one or more biological samples may be obtained,for example, by blood draw, urine collection, biopsy, or other methodsknown in the art. The one or more biological sample may be one or morebiological fluids. The molecule of interest may also be obtained fromspecific organs or tissues, such as muscle, liver, adrenal tissue,prostate tissue, endometrial tissue, blood, skin, and breast tissue.Molecules of interest may be obtained from a specific group of cells,such as tumor cells or fibroblast cells. Molecules of interest also maybe obtained, and optionally partially purified or isolated, from thebiological sample using standard biochemical methods known in the art.

The frequency of biological sampling can vary depending on differentfactors. Such factors include, but are not limited to, the nature of themolecules of interest, ease and safety of sampling, synthesis andbreakdown/removal rates of the molecules of interest, and the half-lifeof a compound (chemical entity, biological factor, already-approveddrug, drug candidate, drug lead, etc.).

The molecules of interest may also be purified partially, or optionally,isolated, by conventional purification methods including high pressureliquid chromatography (HPLC), fast performance liquid chromatography(FPLC), chemical extraction, thin layer chromatography, gaschromatography, gel electrophoresis, and/or other separation methodsknown to those skilled in the art.

In another embodiment, the molecules of interest may be hydrolyzed orotherwise degraded to form smaller molecules. Hydrolysis methods includeany method known in the art, including, but not limited to, chemicalhydrolysis (such as add hydrolysis) and biochemical hydrolysis (such aspeptidase degradation). Hydrolysis or degradation may be conductedeither before or after purification and/or isolation of the molecules ofinterest. The molecules of interest also may be partially purified, oroptionally, isolated, by conventional purification methods includinghigh performance liquid chromatography (HPLC), fast performance liquidchromatography (FPLC), gas chromatography, gel electrophoresis, and/orany other methods of separating chemical and/or biochemical compoundsknown to those skilled in the art.

D. Analysis

Presently available technologies (static methods) used to identifybiological actions of agents measure only composition, structure, orconcentrations of molecules in a cell and do so at one point in time.While RNA and protein expression “chips,” for example, can be used todetect multiple biological molecules at one time in cells or organismsin a variety of disease states, these techniques fail to determine themolecular flux rates of proteins or transcripts. The methods of thepresent invention allow determination of the molecular flux rates of aplurality of proteins or transcripts, as well as the molecular fluxrates of a plurality of organic metabolites, and their changes over timein a variety of disease states and in response to exposure to one ormore drugs, drug candidates, drug leads, or combinations thereof, or inresponse to exposure to one or more industrial chemicals, foodadditives, cosmetics, or environmental pollutants.

1. Mass Spectrometry

Isotopic enrichment biomarkers can be determined by various methods suchas mass spectrometry, including but not limited to gaschromatography-mass spectrometry (GC-MS), isotope-ratio massspectrometry, GC-isotope ratio-combustion-MS, GC-isotoperatio-pyrolysis-MS, liquid chromatography-MS, electrosprayionization-MS, matrix assisted laser desorption-time of flight-MS,Fourier-transform-ion-cyclotron-resonance-MS, and cycloidal-MS.

Mass spectrometers convert molecules into rapidly moving gaseous ionsand separate them on the basis of their mass-to-charge ratios. Thedistributions of isotopes or isotopologues of ions, or ion fragments,may thus be used to measure the isotopic enrichment in a plurality ofproteins or organic metabolites.

Generally, mass spectrometers include an ionization means and a massanalyzer. A number of different types of mass analyzers are known in theart. These include, but are not limited to, magnetic sector analyzers,electrospray ionization, quadrupoles, ion traps, time of flight massanalyzers, and Fourier transform analyzers.

Mass spectrometers may also include a number of different ionizationmethods. These include, but are not limited to, gas phase ionizationsources such as electron impact, chemical ionization, and fieldionization, as well as desorption sources, such as field desorption,fast atom bombardment, matrix assisted laser desorption/ionization, andsurface enhanced laser desorption/ionization.

In addition, two or more mass analyzers may be coupled (MS/MS) first toseparate precursor ions, then to separate and measure gas phase fragmentions. These instruments generate an initial series of ionic fragments ofa protein, and then generate secondary fragments of the initial ions.The resulting overlapping sequences allows complete sequencing of theprotein, by piecing together overlaying “pieces of the puzzle”, based ona single mass spectrometric analysis within a few minutes (plus computeranalysis time).

Different ionization methods are also known in the art. One key advancehas been the development of techniques for ionization of large,non-volatile macromolecules including proteins and polynucleotides.Techniques of this type have included electrospray ionization (ESI) andmatrix assisted laser desorption (MALDI). These have allowed MS to beapplied in combination with powerful sample separation introductiontechniques, such as liquid chromatography and capillary zoneelectrophoresis.

In addition, mass spectrometers may be coupled to separation means suchas gas chromatography (GC) and high performance liquid chromatography(HPLC). In gas-chromatography mass-spectrometry (GC/MS), capillarycolumns from a gas chromatograph are coupled directly to the massspectrometer, optionally using a jet separator. In such an application,the gas chromatography (GC) column separates sample components from thesample gas mixture and the separated components are ionized andchemically analyzed in the mass spectrometer.

When GC/MS (or other mass spectrometric modalities that analyze ions ofbiomolecules, rather than small inorganic gases) is used to measure massisotopomer abundances of organic molecules, hydrogen-labeled isotopeincorporation from isotope-labeled water is amplified 3 to 7-fold,depending on the number of hydrogen atoms incorporated into the organicmolecule from isotope-labeled water in vivo.

In general, in order to determine a baseline mass isotopomer frequencydistribution for a molecule of interest, such a sample is taken beforeinfusion of an isotopically labeled precursor. Such a measurement is onemeans of establishing in the cell, tissue or organism, the naturallyoccurring frequency of mass isotopomers of the molecule of interest.When a cell, tissue or organism is part of a population of subjectshaving similar environmental histories, a population isotopomerfrequency distribution may be used for such a background measurement.Additionally, such a baseline isotopomer frequency distribution may beestimated, using known average natural abundances of isotopes. Forexample, in nature, the natural abundance of ¹³C present in organiccarbon in 1.11%. Methods of determining such isotopomer frequencydistributions are discussed below. Typically, samples of the molecule ofinterest are taken prior to and following administration of anisotopically labeled precursor to the subject and analyzed forisotopomer frequency as described below.

a. Measuring Relative and Absolute Mass Isotopomer Abundances

Measured mass spectral peak heights, or alternatively, the areas underthe peaks, may be expressed as ratios toward the parent (zero massisotope) isotopomer. It is appreciated that any calculation means whichprovide relative and absolute values for the abundances of isotopomersin a sample may be used in describing such data, for the purposes of thepresent invention.

2. Calculating Labeled: Unlabeled Proportion of Molecules of Interest

The proportion of labeled and unlabeled molecules of interest is thencalculated. The practitioner first determines measured excess molarratios for isolated isotopomer species of a molecule. The practitionerthen compares measured internal pattern of excess ratios to thetheoretical patterns. Such theoretical patterns can be calculated usingthe binomial or multinomial distribution relationships as described inU.S. Pat. Nos. 5,338,686, 5,910,403, and 6,010,846, which are herebyincorporated by reference in their entirety. The calculations mayinclude Mass Isotopomer Distribution Analysis (MIDA). Variations of MassIsotopomer Distribution Analysis (MIDA) combinatorial algorithm arediscussed in a number of different sources known to one skilled in theart. The method is further discussed by Hellerstein and Neese (1999), aswell as Chinkes, et al. (1996), and Kelleher and Masterson (1992), andU.S. patent application Ser. No. 10/279,399, all of which are herebyincorporated by reference in their entirety.

In addition to the above-cited references, calculation softwareimplementing the method is publicly available from Professor MarcHellerstein, University of California, Berkeley.

The comparison of excess molar ratios to the theoretical patterns can becarried out using a table generated for a molecule of interest, orgraphically, using determined relationships. From these comparisons, avalue, such as the value p, is determined, which describes theprobability of mass isotopic enrichment of a subunit in a precursorsubunit pool. This enrichment is then used to determine a value, such asthe value A_(X)*, which describes the enrichment of newly synthesizedproteins for each mass isotopomer, to reveal the isotopomer excess ratiowhich would be expected to be present, if all isotopomers were newlysynthesized.

Fractional abundances are then calculated. Fractional abundances ofindividual isotopes (for elements) or mass isotopomers (for molecules)are the fraction of the total abundance represented by that particularisotope or mass isotopomer. This is distinguished from relativeabundance, wherein the most abundant species is given the value 100 andall other species are normalized relative to 100 and expressed aspercent relative abundance. For a mass isotopomer M_(X),

${{{Fractional}\mspace{14mu}{abundance}\mspace{14mu}{of}\mspace{14mu} M_{X}} = {A_{X} = \frac{{Abundance}\mspace{14mu} M_{x}}{\sum\limits_{i = 0}^{n}{{Abundance}\mspace{14mu} M_{i}}}}},$where 0 to n is the range of nominal masses relative to the lowest mass(M₀) mass isotopomer in which abundances occur.

${{\Delta\mspace{14mu}{Fractional}\mspace{14mu}{abundance}\mspace{14mu}\left( {{enrichment}\mspace{14mu}{or}\mspace{14mu}{depletion}} \right)} = {{\left( A_{x} \right)_{e} - \left( A_{x} \right)_{b}} = {\left( \frac{{Abundance}\mspace{14mu} M_{x}}{\sum\limits_{i = 0}^{n}{{Abundance}\mspace{14mu} M_{i}}} \right)_{e} - \left( \frac{{Abundance}\mspace{14mu} M_{x}}{\sum\limits_{i = 0}^{n}{{Abundance}\mspace{14mu} M_{i}}} \right)_{b}}}},$

where subscript e refers to enriched and b refers to baseline or naturalabundance.

In order to determine the fraction of polymers that were actually newlysynthesized during a period of precursor administration, the measuredexcess molar ratio (EM_(X)) is compared to the calculated enrichmentvalue, A_(X)*, which describes the enrichment of newly synthesizedbiopolymers for each mass isotopomer, to reveal the isotopomer excessratio which would be expected to be present, if all isotopomers werenewly synthesized.

3. Calculating Molecular Flux Rates

The method of determining rate of synthesis includes calculating theproportion of mass isotopically labeled subunit present in the molecularprecursor pool, and using this proportion to calculate an expectedfrequency of a molecule of interest containing at least one massisotopically labeled subunit. This expected frequency is then comparedto the actual, experimentally determined isotopomer frequency of themolecule of interest. From these values, the proportion of the moleculeof interest which is synthesized from added isotopically labeledprecursors during a selected incorporation period can be determined.Thus, the rate of synthesis during such a time period is alsodetermined.

A precursor-product relationship may then be applied. For the continuouslabeling method, the isotopic enrichment is compared to asymptotic(i.e., maximal possible) enrichment and kinetic parameters (e.g.,synthesis rates) are calculated from precursor-product equations. Thefractional synthesis rate (k_(s)) may be determined by applying thecontinuous labeling, precursor-product formula:k _(s)=[−ln(1−f)]/t,

where f=fractional synthesis=product enrichment/asymptoticprecursor/enrichment

and t=time of label administration of contacting in the system studied.

For the discontinuous labeling method, the rate of decline in isotopeenrichment is calculated and the kinetic parameters of the molecules ofinterest are calculated from exponential decay equations. In practicingthe method, biopolymers are enriched in mass isotopomers, preferablycontaining multiple mass isotopically labeled precursors. These highermass isotopomers of the molecules of interest, e.g., moleculescontaining 3 or 4 mass isotopically labeled precursors, are formed innegligible amounts in the absence of exogenous precursor, due to therelatively low abundance of natural mass isotopically labeled precursor,but are formed in significant amounts during the period of molecularprecursor incorporation. The molecules of interest taken from the cell,tissue, or organism at the sequential time points are analyzed by massspectrometry, to determine the relative frequencies of a high massisotopomer. Since the high mass isotopomer is synthesized almostexclusively before the first time point, its decay between the two timepoints provides a direct measure of the rate of decay of the molecule ofinterest.

Preferably, the first time point is at least 2-3 hours afteradministration of precursor has ceased, depending on mode ofadministration, to ensure that the proportion of mass isotopicallylabeled subunit has decayed substantially from its highest levelfollowing precursor administration. In one embodiment, the followingtime points are typically 1-4 hours after the first time point, but thistiming will depend upon the replacement rate of the biopolymer pool.

The rate of decay of the molecule of interest is determined from thedecay curve for the three-isotope molecule of interest. In the presentcase, where the decay curve is defined by several time points, the decaykinetic can be determined by fitting the curve to an exponential decaycurve, and from this, determining a decay constant.

Breakdown rate constants (k_(d)) may be calculated based on anexponential or other kinetic decay curve:k _(d)=[−ln f]/t.

As described, the method can be used to determine subunit poolcomposition and rates of synthesis and decay for substantially anybiopolymer which is formed from two or more identical subunits which canbe mass isotopically labeled. Other well-known calculation techniquesand experimental labeling or de-labeling approaches can be used (e.g.,see Wolfe, R. R. Radioactive and Stable Isotope Tracers in Biomedicine:Principles and Practice of Kinetic Analysis. John Wiley & Sons; (March1992)) for calculation flux rates of molecules and flux rates throughmetabolic pathways of interest.

E. Uses of the Methods of the Present Invention

The disclosed invention allows for the measurement of molecular fluxeswithin metabolic pathways or networks that are believed to be a drivingfactor for, or etiologic mechanism of, a disease of interest. Molecularfluxes through the metabolic pathway or network itself is the biomarkerfor analysis, as it fundamentally represents the physiological andpathophysiological process of the living system. By using the methods ofthe present invention, one can quantitate the molecular flux rates ofone or more molecules of interest within one or more targeted metabolicpathways or networks and use the information as a biomarker of medicaldiagnosis, prognosis, or therapeutic activity of drug or combinationdrug treatment. The methods allow for the characterization or evaluation(or both the characterization and evaluation) of compounds and enableone of skill to assess therapeutic efficacy and/or toxic effects.

The methods disclosed herein allow for effects on biomarkers to beobserved after a living system is exposed to a compound or combinationsof compounds. The data generated and analyzed is therefore useful in theDDA process as it facilitates the DDA decision-making process; i.e., itprovides useful information for decision-makers in their decision tocontinue with further development on a compound or combination ofcompounds (e.g., if the biomarker data appear promising) or to ceasesaid efforts, for example, if the biomarker data appear unfavorable (seeFIG. 1 for a graphical depiction of this process).

Moreover, the methods allow for the skilled artisan to identify, select,and/or characterize “best in breed” in a class of compounds. Onceidentified, selected, and/or characterized, the skilled artisan, basedon the information generated by the methods of the present invention,can decide to evaluate the “best in breed” further or to license thecompound to another entity such as a pharmaceutical company orbiotechnology company.

In another embodiment, the methods of the present invention allow forthe characterization or evaluation (or both the characterization andevaluation) of toxic effects from exposure to industrial chemicals, foodadditives, cosmetics, and environmental pollutants. The methods of thepresent invention can be used to establish programs to identify andexplore the molecular mechanisms of industrial, food, cosmetic, andenvironmental toxicants to further public health goals.

In one embodiment, the molecular flux rates in the one or more metabolicpathways being measured may be relevant to an underlying molecularpathogenesis, or causation of, one or more diseases. In anotherembodiment, the molecular flux rates in one or more metabolic pathwaysof interest may contribute to the initiation, progression, severity,pathology, aggressiveness, grade, activity, disability, mortality,morbidity, disease sub-classification or other underlying pathogenic orpathologic feature of the disease of interest.

In yet another embodiment, the molecular flux rates in one or moremetabolic pathways of interest may contribute to the prognosis,survival, morbidity, mortality, stage, therapeutic response,symptomology, disability or other clinical factor of the disease ofinterest. Two or more molecular flux rates in metabolic pathways may bemeasured independently or concurrently.

Such metabolic pathways may include, but are not limited to, hepatocyteproliferation and destruction (or inhibition of proliferation), totalliver cell proliferation and destruction (or inhibition ofproliferation), renal tubular cell turnover, lymphocyte turnover,spermatocyte turnover, protein synthesis and breakdown in muscle andheart, liver collagen synthesis and breakdown, myelin synthesis andbreakdown in brain or peripheral nerves, neuron proliferation anddestruction (or inhibition of proliferation), neuroprogenitor cellproliferation and destruction (or inhibition of proliferation), breastepithelial cell proliferation and destruction (or inhibition ofproliferation), colon epithelial cell proliferation and destruction (orinhibition of proliferation), prostate epithelial cell proliferation anddestruction (or inhibition of proliferation), ovarian epithelial cellproliferation and destruction (or inhibition of proliferation),endometrial cell proliferation and destruction (or inhibition ofproliferation), endothelial cell proliferation and destruction (orinhibition of proliferation), bronchial epithelial cell proliferationand destruction (or inhibition of proliferation), pancreatic epithelialcell proliferation and destruction (or inhibition of proliferation),pancreatic β cell proliferation, pancreatic islets of Langerhansproliferation and destruction (or inhibition of proliferation),microglia proliferation and destruction (or inhibition), keratinsynthesis in skin, keratinocyte proliferation and destruction (orinhibition of proliferation), immunoglobulin synthesis and breakdownincluding M protein synthesis and breakdown, synthesis and breakdown ofmitochondrial DNA, synthesis and breakdown of mitochondrialphospholipids, DNA methylation and demethylation, synthesis andbreakdown of mitochondrial proteins, synthesis and breakdown of adiposelipids, and synthesis and breakdown of adipose cells.

Known animal models of disease may be used as part of the presentinvention. Such animal models of disease may include, but are notlimited to, Alzheimer's disease, heart failure, renal disease, diabeticnephropathy, osteoporosis, hepatic fibrosis, cirrhosis, hepatocellularnecrosis, pulmonary fibrosis, scleroderma, renal fibrosis, multiplesclerosis, arteriosclerosis (or atherosclerosis), osteoarthritis,rheumatoid arthritis, psoriasis, skin photoaging, skin rashes, breastcancer, prostate cancer, colon cancer, pancreatic cancer, lung cancer,acquired immunodeficiency syndrome, immune defects, multiple myeloma,chronic lymphocytic leukemia, chronic myelocytic leukemia, diabetes,diabetic complications, insulin resistance, obesity, lipodystrophy,metabolic syndrome (or syndrome X), muscle wasting, frailty,deconditioning, angiogenesis, hyperlipidemia, infertility, viral orbacterial infections, auto-immune disorders, and immune flares.

These interactions between compounds cannot be detected or quantified byuse of contemporary or traditional assays that investigate one moleculartarget and step at a time in a disease-related pathway. A method forsystematically evaluating compounds including chemical entities,biologics, combinations of chemical entities, or combinations ofbiologics for effects on molecular fluxes through pathways had notpreviously been available. The invention disclosed herein wouldfacilitate the process of identifying, developing and approvingeffective therapeutic combinations of drug agents.

In another embodiment, the methods of the invention are useful indetecting toxic effects of industrial or occupational chemicals, foodadditives, cosmetics, or environmental pollutants/contaminants. Suchtoxic effects may include end-organ toxicity. End-organ toxicity mayinclude, but is not limited to, hepatocyte proliferation and destruction(or inhibition of proliferation), total liver cell proliferation anddestruction (or inhibition of proliferation), renal tubular cellturnover, lymphocyte turnover, spermatocyte turnover, protein synthesisand breakdown in muscle and heart, liver collagen synthesis andbreakdown, myelin synthesis and breakdown in brain or peripheral nerves,neuron proliferation and destruction (or inhibition of proliferation),neuroprogenitor cell proliferation and destruction (or inhibition ofproliferation), breast epithelial cell proliferation and destruction (orinhibition of proliferation), colon epithelial cell proliferation anddestruction (or inhibition of proliferation), prostate epithelial cellproliferation and destruction (or inhibition of proliferation), ovarianepithelial cell proliferation and destruction (or inhibition ofproliferation), endometrial cell proliferation and destruction (orinhibition of proliferation), endothelial cell proliferation anddestruction (or inhibition of proliferation), bronchial epithelial cellproliferation and destruction (or inhibition of proliferation),pancreatic epithelial cell proliferation and destruction (or inhibitionof proliferation), pancreatic β cell proliferation, pancreatic islets ofLangerhans proliferation and destruction (or inhibition ofproliferation), microglia proliferation and destruction (or inhibition),keratin synthesis in skin, keratinocyte proliferation and destruction(or inhibition of proliferation), immunoglobulin synthesis and breakdownincluding M protein synthesis and breakdown, synthesis and breakdown ofmitochondrial DNA, synthesis and breakdown of mitochondrialphospholipids, DNA methylation and demethylation, synthesis andbreakdown of mitochondrial proteins, synthesis and breakdown of adiposelipids, and synthesis and breakdown of adipose cells.

FIG. 6 illustrates the use of the inventions herein in a drug discoveryprocess. At step 601 a plurality of drug candidates or other compoundsare selected. At step 603 the flux rates of biomarkers are studiedwithin cells, preferably according to the methods discussed herein. Inalternative embodiments, step 603 is conducted first when the inventionsare used, for example, in a target discovery process. At step 605relevant flux rates are identified. For example, if it is desirable toreduce the flux rate of a particular biomarker in a particularphenotypic state, a compound that reduces that flux rate will beconsidered generally more useful, and conversely a compound thatincreases that flux rate will be considered generally less desirable. Ina target discovery process, a particular phenotype that has increased ordecreased flux rates with respect to another phenotype (e.g., diseasedvs. not diseased) may be considered a good therapeutic or diagnostictarget or in the pathway of a good therapeutic or diagnostic target. Atstep 607 compounds of interest, targets of interest, or diagnostics areselected and further used and further developed. In the case of targets,such targets may be the subject of, for example, well known smallmolecule screening processes (e.g., high-throughput screening of newchemical entities) and the like. Alternatively, biological factors, oralready-approved drugs, or other compounds (or combinations and/ormixtures of compounds) may be used. At step 609 the compounds ordiagnostics are sold or distributed. It is recognized of course that oneor more of the steps in the process in FIG. 6 will be repeated manytimes in most cases for optimal results.

Table 1 depicts examples of biomarkers, the related clinical or medicaldiseases or conditions and the molecule of interest to be detected usingthe methods of the application. Taking into account Table 1, the presentapplication is further directed to a method for monitoring or diagnosinga clinical or medical disease or condition, the method including: a)administering an isotope-labeled substrate to the living system for aperiod of time sufficient for the isotope-labeled substrate to enterinto one or more metabolic pathways of interest and thereby enter intoand label at least one targeted molecule of interest within the one ormore metabolic pathways of interest in the living system; b) obtainingone or more samples from the living system, wherein the one or moresamples include at least one isotope-labeled targeted molecule ofinterest; c) measuring the content, rate of incorporation and/or patternor rate of change in content and/or pattern of isotope labeling of theat least one targeted molecule of interest; d) calculating molecularflux rates in the one or more metabolic pathways/biomarker of interestbased on the content and/or pattern or rate of change of content and/orpattern of isotopic labeling in the at least one targeted molecule ofinterest to monitor or diagnose the clinical or medical disease orcondition. In another format, one or more compounds are administered tothe living system before or after the determination of the molecularflux rates of the one or more metabolic pathways of interest in theliving system in order to evaluate the effect of the one or morecompounds on the biomarker as a predictor of an effect of the compoundon the clinical or medical disease or condition.

TABLE 1 Dynamic Biomarkers and Applications Physiological/ Clinical orMedical Medical Area Biomarker Disease or Condition Molecule of interestMetabolism/ adipose triglyceride Obesity; lipoatrophy; Glycerol or fattyNutrition/ dynamics fat distribution; acids derived from Endocrinologyhyperplasia- triglyceride. hypertrophy Adipocyte dynamics hyperplasia-DNA isolated from hypertrophy; response adipocytes to compounds ortherapeutics. Muscle mitochondrial Unfitness; DNA from muscle DNA orphospholipid cardiovascular disease mitochondria or dynamics risk;autotoxicity phospholipids (e.g., drugs; deconditioning; cardiolipin)from frailty muscle mitochondria. Muscle protein Frailty; wasting;Protein derived dynamics sports; dystrophies from muscle (e.g., totalmuscle protein or myosin) Dynamics of adipose Atherosclerosis; Glycerolor fatty lipolysis diabetes mellitus risk acids derived fromtriglyceride. Dynamics of adipose or carbohydrate Fatty acids hepaticoverfeeding; anabolic de novo lipogenesis block or impaired fatoxidation; energy balance Dynamics of glycolysis Insulin resistance;Water (whole body, increase impaired glucose or decrease) tolerance;diabetes mellitus risk Dynamics of metabolic Obesity risk; hypo or WaterH₂O or CO₂ production hypermetabolism; (whole body) thermogenic;response to compounds or therapeutics Dynamics of fatty acid Obesityrisk; fuel Water oxidation selector; insulin resistance; Dynamics ofhepatic Hepatic insulin Glucose glucose production resistance; hypo or(endogenous glucose hypermetabolism production) Dynamics of hepaticHepatic steatosis Glycerol or fatty triglyceride synthesis (tumors;cirrhosis) acids derived from (de novo lipogeneis + triglyceride, fattysecretion) acids β-Cell DNA dynamics Pancreatic burden/ DNA derived frompancreatic reserve; pancreatic beta diabetes mellitus risk cells.Insulin dynamics Pancreatic burden/ Insulin pancreatic reserve advancedglycation Diabetes mellitus Advanced glycation endproduct dynamics;complications endproducts advanced glycation (glycated proteins).endproduct glycosylation dynamics Keratinocyte or Caloric DNA derivedfrom mammary epithelial cell restriction/longevity keratinocytes ordynamics regiments mammary epithelial cells. Hepatic bile acidhyperlipoproteinemia Hepatic bile acids dynamics Dynamics of conversionhyperlipoproteinemia; Fatty acids and of ethanol to acetatecirrhosis/steatosis risk acetate and triglyceride CardiovascularApolipoprotein B Coronary artery Apolipoprotein B dynamics disease riskVery low density Coronary artery Apolipoprotein B, lipoprotein (VLDL) -disease risk; Triglyceride, tryglyceride dynamics pancreatitis; glycerolhyperlipoproteinemia Hepatic + whole body Statin response; Cholesterolfrom Cholesterol dynamics Coronary artery serum/blood or disease risk;diet other biological treatment sample Vascular smooth Atherosclerosisrisk DNA derived from muscle cell dynamics vascular smooth muscle cellsCholesterol transport Coronary artery Bile acids, dynamics (reversedisease risk Cholesterol cholesterol transport) Cardiac muscle proteinCardiomyopathy Protein derived dynamics from cardiac muscle (e.g., totalprotein or myosin) Cardiac collagen Cardiac fitness, Collagen (e.g.,type dynamics congestive heart III collagen) failure derived fromcardiac tissue Vascular smooth Vasculitis DNA derived from muscle cellor vascular smooth endothelial cell muscle cells or dynamics endothelialcells Skeletal/ Keratinocyte dynamics Psoriasis; skin DNA derived fromRheumatic/ hyperproliferation; keratinocytes Integument ectopy Skinkeratin dynamics Psoriasis, skin barrier Skin keratin (Type I and/ortype II) Skin collagen dynamics + Skin wrinkles; Collagen (e.g., typeelastin dynamics dermatomyolitis; III collagen) from scleroderma skin(epidermis or dermis) Wound collagen Wound healing; Collagen (e.g., typedynamics adjunctive compound III collagen) from or therapeutic skin andother response wounded tissues Synovial space osteoarthritis; Hyaluronicacid hyaluronic acid or rheumatoid arthritis; from synovial fluidchondroitin sulfate joint or cartilage, dynamics protection/destruction;chondroitin sulfate diet or compound or from synovial fluid therapeuticresponse or cartilage Bone collagen Osteoporosis; pagets; Collagen(e.g., type dynamics healing of bone I collagen) from fractures boneJoint collagen dynamics osteoarthritis; Collagen (e.g., type rheumatoidarthritis; II collagen) from joint protection; synovial fluid orresponse to treatment cartilage Synovial leukocyte/T- rheumatoidarthritis; DNA from Cell dynamics joint destruction leukocytes or T-cells in synovial fluid or associated with joints Oncology/ Mammaryepithelial cell Risk for cancer; DNA from Neoplasia dynamics compound ormammary epithelial therapeutic response cells Colon epithelial cell Riskfor cancer; DNA from colon dynamics compound or epithelial cellstherapeutic response Bronchial cell or tissue Risk for cancer; DNA frombronchial dynamics compound or tissue therapeutic response Prostateepithelial cell Risk for cancer; benign DNA from prostate dynamicsprostate hyperplasia; epithelial cells compound or therapeutic responseDynamics of tumors of Risk for cancer; DNA from cells from pancreas,bladder, compound or which tumors may gastric, brain, ovary, therapeuticresponse derive (e.g., cervix epithelial cells) or pre-cancerous cells,or cells whose proliferative behavior is associated with increased riskof cancer Dynamics of solid Tumor growth; grade; DNA derived from tumors(including prognosis; solid tumor cells breast, colon, lung,aggressiveness; Rx lymphoma) response Dynamics of liquid Cancer growth,DNA derived from tumors prognosis, compound liquid tumor cells ortherapeutic response Immunoglobulin, Multiple myeloma Myeloma protein,albumin, or myeloma- activity, prognosis, immunoglobulins, proteindynamics. growth, mass, or albumin derived Myeloma cell dynamicscompound or from serum or bone therapeutic response marrow, DNA frommyeloma cells. Tumor endothelial cell Angiogenesis; DNA from tumordynamics compound or endothelial cells therapeutic response Dynamics ofcompound or Deoxyadenosine ribonucleotide therapeutic response anddeoxythymidine reductase substrates and metabolites (flux vs. salvage)Epithelial stem cell Cancer risk; compound DNA from epithelial dynamicsor therapeutic stem cells response Tumor cell RNA Tumor grade; RNA fromtumor dynamics prognosis; treatment cells, total or target; compound ortranscript specific therapeutic response T-cell or other bloodProliferation + growth DNA from cell dynamics (post of transplanttransplanted cells, bone marrow or from cells transplant) maturing fromtransplanted cells Cell dynamics at Adequacy of surgery DNA from thesurgical margin of surgical margin of tumor the tumor Grafted tissueGrade, aggressiveness; DNA from the dynamics graft-versus-host- graftedtissue disease treatment response Dynamics of Gene silencing; Methylmethylcytosine, (methyl prognosis; compound deoxycytosine fromdeoxycytosine or therapeutic DNA from cells of methylation/ responseinterest hypomethylation) Dynamics of histone Gene expression; Histonesfrom cells acetylation and prognosis; histone of interest deacetylationacetylation; compound or therapeutic response Neurology Brain Amyloid-βor Alzheimer's disease Amyloid beta amyloid precursor risk; response topeptide or amyloid protein dynamics treatment precursor protein orsubfragments of either Brain or peripheral Multiple sclerosis (MS)Galactocerebroside nervous system myelin activity; MS response frombrain, dynamics to treatment; spinal peripheral nervous cord + braininjury system, or blood recovery and/or compound or therapeutic responseNeuron dynamics Neurogenesis; learning DNA from neurons x-ray therapytoxicity; development; stress; depression Neurotransmitter Psychiatricdisorders Neurotransmitters dynamics from brain or PNS (e.g., serotonin,dopamine, glutamate), or circulating or degraded neurotransmitters foundin other tissues Neuroprogenitor cell Neurogenesis, DNA from dynamicsdepression, compound neuroprogenitor or therapeutic cells responseMicroglial cell dynamics Neuroinflammation, DNA from microglia multiplesclerosis, Alzheimer's disease, stroke, autism, depression, chronicpain, amyotrophic lateral sclerosis, cerebral amyloid angiopathy,excitotoxic injury, compound or therapeutic response Brain microtubuleMemory, learning, Microtubules dynamics alzheimer's disease, (tubulin)from excitotoxic injury, central or peripheral neurogenesis, nervoussystem, neurodegenerative microtubule diseases, compound or subfractions(e.g., therapeutic response tau-associated, dimeric, polymeric)Gastrointestinal/ Hepatocyte dynamics Hepatic necrosis; toxin DNA fromOther Internal exposure; hepatitis; hepatocytes Organs response totreatment Hepatic collagen Hepatic fibrosis, Collagen (e.g. typedynamics cirrhosis risk, I or III collagen) prognosis, disease fromliver activity, response to treatment Hepatic mitochondrial Effects fromexposure DNA or dynamics to hepatic toxins, phospholipids (e.g.mitochondrial toxins, cardiolipin) from recovery, response to hepatictreatment mitochondria Renal epithelial cell Effects from exposure DNAfrom renal dynamics to nephrotoxins, epithelial cells. recovery,response to treatment Renal collagen DM nephropathy risk Collagen (e.g.type dynamics and activity, response III collagen) from to treatmentkidney Pulmonary collagen Pulmonary fibrosis Pulmonary (e.g. dynamicsdisease activity, type III collagen) response to treatment; collagenblack lung; hypersensitivity pneumonitis; asbestosis; silicosis; chronicobstructive pulmonary disease Pulmonary elastin Emphysema: Pulmonaryelastin dynamics prognosis, compound or therapeutic response ColonocyteDNA Inflammatory bowel DNA from dynamics disease: activity, colonocytesisolated prognosis, compound from stool, colon or therapeutic biopsy, orother response colon tissue sample Gastric epithelial DNA H. pyloriactivity, DNA from gastric dynamics compound or epithelial cellstherapeutic response; cancer risk; gastric cancer Immunologic/ T-celldynamics Cell mediated DNA from T-cells Inflammatory immunity; immuneactivation; AIDS, compound or therapeutic response Antigen-specificT-cell Vaccination response; DNA from T-cells dynamics host defense vs.isolated based on pathogen; adjunctive their antigen compound orspecificity therapeutic response Naïve T-Cell dynamics Thymopoiesis;thymic DNA from naïve T- failure; compound or cells therapeutic responseSpecific antibody B-cell/plasma cell: Immunoglubulin dynamics activity,compound or (e.g., IgG) specific therapeutic response, to antigen ofchoice vaccine response Serum acute-phase Immune activation, Acute phasereactant dynamics disease activity proteins (e.g., c- reactive protein)Plasma cell dynamics Humoral immunity DNA from plasma cells Naturalkiller cell Host defense activity, DNA from natural dynamics compound orkiller cells therapeutic response (e.g., interleukin-2) Cytokinedynamics Endogenous response Secreted or tissue to exogenous associatedcompound or cytokines (e.g., therapeutic; host interleukin-1, defenseinterleukin-2, tumor necrosis factor alpha) Infectious Viral DNA/RNAViral replication, DNA or RNA from dynamics (e.g., HIV, diseaseactivity, virus of interest Hepatitis B) compound or therapeuticresponse, sensitivity to antiviral agents Viral protein dynamics Viralreplication, Protein from virus disease activity, of interest compoundor therapeutic response, sensitivity to antiviral agents Bacterialdynamics Bacterial cell division; DNA or other disease activity;molecule (e.g., response to antibiotics protein, carbohydrate, lipid)from bacteria of interest Parasite dynamics Parasite division and DNA orother growth; compound or molecule (e.g., therapeutic response protein,(e.g., malaria, carbohydrate, lipid) schistosomiasis) from parasite ofinterest Intestinal microbial Infectious activity; DNA or other dynamicscompound or molecule (e.g., therapeutic response protein, carbohydrate,lipid) from intestinal bacteria Bacterial dynamics Abscess; empyema;Bacterial DNA or (closed space) compound or other molecule therapeuticresponse (e.g., protein, carbohydrate, lipid) from tissue or abscess orfluid sample Endovascular bacterial Endocarditis compound DNA or otherdynamics or therapeutic molecule (e.g., response protein, carbohydrate,lipid) from endovascular bacteria Hematologic Bone marrow precursor Stemcell response DNA from bone cell dynamics. Bone (transplant, compoundmarrow precursor marrow cell dynamics. or therapeutic); status cells.DNA from of cytopenias bone marrow cells. hemoglobin dynamics Hemolysis;anemia Hemoglobin (red blood cells) response (reticulocytosis);hemoglobinopathies Platelet phospholipid or Thrombocytopenia;Phospholipids or dynamics thrombocytosis DNA from platelets or plateletprecursors Erythrocyte membrane Anemia; hemolysis; Phospholipids fromdynamics compound or erythrocytes therapeutic response Genetic/Spermatocyte dynamics Spermatogenesis; DNA from Developmental/ maleinfertility; spermatocytes Reproductive compound or therapeuticresponse; endocrine disruptors Timing of embryonic Developmental biologyEmbryonic proteins, protein, lipid, and and disorders thereof lipids, orDNA dynamics Genomic DNA dynamics Genetic instability; Genomic DNA (fromcancer risk at risk tissue if appropriate) Table abbreviations: DNA =deoxyribonucleic acid.

F. Isotopically-Perturbed Molecules

In another variation, the methods provide for the production ofisotopically-perturbed molecules (e.g., labeled fatty acids, lipids,carbohydrates, proteins, nucleic acids and the like) or fragments ordegradatory products thereof. These isotopically-perturbed molecules orfragments or degradatory products thereof comprise information useful indetermining the flux of molecules within the metabolic pathways ofinterest. Once isolated from a cell and/or a tissue of an organism, oneor more isotopically-perturbed molecules or fragments or degradatoryproducts thereof are analyzed to extract information as described,supra.

G. Kits

The invention also provides kits for measuring and comparing molecularflux rates in vivo. The kits may include isotope-labeled precursormolecules, and may additionally include chemical compounds known in theart for separating, purifying, or isolating proteins, and/or chemicalsnecessary to obtain a tissue sample, automated calculation software forcombinatorial analysis, and instructions for use of the kit.

Other kit components, such as tools for administration of water (e.g.,measuring cup, needles, syringes, pipettes, IV tubing), may optionallybe provided in the kit. Similarly, instruments for obtaining samplesfrom the cell, tissue, or organism (e.g., specimen cups, needles,syringes, and tissue sampling devices) may also be optionally provided.

H. Information Storage Devices

The invention also provides for information storage devices such aspaper reports or data storage devices comprising data collected from themethods of the present invention. An information storage deviceincludes, but is not limited to, written reports on paper or similartangible medium, written reports on plastic transparency sheets ormicrofiche, and data stored on optical or magnetic media (e.g., compactdiscs, digital video discs, optical discs, magnetic discs, and thelike), or computers storing the information whether temporarily orpermanently. The data may be at least partially contained within acomputer and may be in the form of an electronic mail message orattached to an electronic mail message as a separate electronic file.The data within the information storage devices may be “raw” (i.e.,collected but unanalyzed), partially analyzed, or completely analyzed.Data analysis may be by way of computer or some other automated deviceor may be done manually. The information storage device may be used todownload the data onto a separate data storage system (e.g., computer,hand-held computer, and the like) for further analysis or for display orboth. Alternatively, the data within the information storage device maybe printed onto paper, plastic transparency sheets, or other similartangible medium for further analysis or for display or both.

I. Examples

The following non-limiting examples further illustrate the inventiondisclosed herein:

Example 1: Triglyceride Synthesis (Lipogenesis) and Breakdown(Lipolysis) in Rats as a Biomarker of Obesity-Related Diseases orConditions

Triglyceride synthesis is the fundamental biochemical process (i.e.,metabolic pathway) for fat formation (lipogenesis) and therefore is abiomarker for obesity-related diseases or conditions (obesity itself isa condition and is the principal, but not the only, condition ofinterest herein). Determining whether a compound or a combination ofcompounds or a mixture of compounds (e.g., a chemical entity such as anew chemical entity (NCE), or combinations of chemical entities such asa combination of NCEs, drug candidate, or a combination of drugcandidates, drug lead, or a combination of drug leads, or analready-approved drug such as one listed in the Physician's DeskReference (PDR) or Merck Index, or a combination of already-approveddrugs, or a biological factor, or a combination of biological factors(or any combination of mixtures of NCEs, drug candidates, drug leads,already-approved drugs, and/or biological factors) can inhibitlipogenesis is important in determining whether a compound, orcombination of compounds, or mixture of compounds has potential fortreating obesity-related diseases or conditions or other metabolicdisorders.

To assess whether a compound, or a combination of compounds, or amixture of compounds inhibits lipogenesis (and therefore, as statedabove, a candidate drug specific for treating obesity-related diseasesor conditions, or other metabolic disorders) Sprague-Dawley rats(200-300 g Simonsen Labs, Gilroy, Calif.) are either exposed to acompound, or combination of compounds, or mixture of compounds, or leftunexposed (i.e., controls). Rats are administered a compound orcombination of compounds or a mixture of compounds or vehicle via anappropriate route of administration. One compound, or a combination ofcompounds, or a mixture of compounds may be administered.

An initial priming dose of 99.8% ²H₂O is given via intraperitonealinjection to achieve ca. 2.5% body water enrichment (assuming 60% bodyweight as water) followed by administration of 8% ²H₂O in drinking waterfor up to 12 weeks.

Adipose tissue samples are placed in dual glass tissue grinders (e.g.,Kontes tissue grinders, Kimble Kontes, Vineland, N.J.) with 1 mlmethanol:chloroform (2:1), ground until homogenous then centrifuged toremove protein. The solution is extracted with 2 ml each chloroform andwater. The aqueous phase is discarded and the lipid fraction istransesterified by incubation with 3N methanolic HCL (Sigma-Aldrich) at55° C. for 60 min. Fatty acid methyl esters are separated from glycerolby the Folch technique, with the modification that pure water ratherthan 5% NaCl is used for the aqueous phase. The aqueous phase containingglycerol is then lyophilized and glycerol is converted to glyceroltri-acetate by incubation with acetic anhydride:pyridine, 2:1 asdescribed elsewhere (Hellerstein, M. K., R. A. Neese, and J. M. Schwarz.Am J Physiol 265:E814-20, 1993, herein incorporated by reference). Somesamples are extracted and then TG separated from other acylglycerides bythin layer chromatography (TLC) as described elsewhere (Jung, H. R., S.M. Turner, R. A. Neese, S. G. Young, and M. K. Hellerstein. Biochem J343 Pt 2:473-8, 1999, herein incorporated by reference), then analyzedas described, supra.

Glycerol-triacetate is analyzed for isotope enrichment by GC/MS, asdescribed, supra.

The fraction of TG that is newly synthesized, (f) is calculated asdescribed, supra.

The theoretical plateau or asymptotic value (A₁ ^(∞)) in TG-glycerolduring ²H₂O labeling is determined in two ways: by mass isotopomerdistribution analysis (MIDA) of the combinatorial labeling pattern inglycerol (A₁ ^(∞) _(mida)) and by measurement of plateau enrichmentsreached in “fully replaced” TG depots (A₁ ^(∞) plateau) (see below). Thestandard precursor-product equation is then applied:f=1−e−ks*tks=−ln(1−f)/t

Where ks represents the fractional replacement or synthesis rateconstant and t is time of labeling.

The absolute synthesis rate of adipose TG is calculated by multiplyingthe measured fractional synthesis (ks) over the period of labeling timesthe pool size of TG. For the purpose of this calculation, TG content isassumed to be 10% of body weight in non-obese young rodents. Theabsolute synthesis rate of adipose tissue TG can be calculated asfollows,Absolute synthesis (mg/d)=ks(d−1)×TG content (mg)

For statistical analysis, ANOVA is used to compare groups with p<0.05 asthe criteria for significance. Curve fitting of label incorporation datais performed using Delta Graph (Delta Point, Inc.).

TG synthesis rates are then compared between exposed animals andunexposed animals to determine whether a compound, or a combination ofcompounds, or a mixture of compounds inhibits lipogenesis, whichprovides a basis for selecting compounds, combinations of compounds, ormixtures of compounds for development and evaluation for treating suchobesity-related diseases and conditions.

One can also assess whether a compound, or a combination of compounds,or a mixture of compounds stimulates lipolysis using the protocols asdescribed, supra. Stimulating lipolysis is also important in treatingobesity-related diseases and conditions or other metabolic disorders;therefore, determining whether a compound, or a combination ofcompounds, or a mixture of compounds stimulates lipolysis is importantin determining whether the compound, or combination of compounds, ormixture of compounds has potential to treat obesity-related diseases orconditions.

The net lipolytic (TG breakdown) rate in individual fat depots iscalculated from the difference between the absolute rate of TG synthesisand the net rate of TG accumulation, where the latter is determined fromthe change in weight over time in a fat pad or in the whole body:

Net  lipolysis  (mg/d) = Absolute  TG  synthesis − net  TG  accumlation = ([ks(d − 1) × TG  content  (mg)] − [(change  in  TG  content)/time  (d)])

Exposed animals are then compared to unexposed animals to determine if acompound or a combination of compounds or a mixture of compounds haslipolytic activity, which provides a basis for selecting and/orcharacterizing compounds for development and evaluation in treatingindications such as obesity-related diseases and conditions and forevaluating efficacy, dose, etc.

As shown in FIG. 21, adipose lipolysis and triglyceride synthesis areincreased in obese mice, which can be suppressed by leptinadministration.

Example 2: DNA Synthesis in Rats as a Biomarker of Cell Proliferation

DNA synthesis is the fundamental biochemical process (i.e., metabolicpathway) for cell proliferation and is therefore a biomarker for cellproliferation. In some settings it may be desirable to stimulate cellproliferation (e.g., wound healing) while in other settings it may bedesirable to inhibit cell proliferation (e.g., cancer).

Rats are administered ²H₂O as discussed in Example 1, supra. DNA islabeled by ²H as shown in FIGS. 2 and 3.

Rats are administered a compound or a combination of compounds or amixture of compounds or vehicle (controls) as discussed in Example 1,supra.

DNA is then isolated from the tissue or cell of interest using a Qiagenkit (Qiagen, Valencia, Calif.), following the manufacturer's protocol.Isolated DNA, eluted in water, is adjusted to pH 9-10 and hydrolyzedenzymatically; deoxyribose is released selectively from purine (dA/dG)deoxynucleotides and converted to the pentane tetraacetate derivative.Alternatively, a pentafluorobenzyl derivative is prepared by reactionwith excess pentafluorobenzyl hydroxylamine under acidic conditions,followed by acetylation with acetic anhydride. Either type of derivativeis subsequently extracted with an organic solvent, dried with sodiumsulfate, and analyzed by GC/MS as described, supra.

Isotope enrichment is then analyzed and flux rates calculated asdescribed, supra. DNA synthesis is then determined as described, supra,and in U.S. Pat. No. 5,910,403. Exposed animals are then compared tounexposed animals to determine if the compound or combination ofcompounds or mixture of compounds has an effect on DNA synthesis (i.e.,stimulation or inhibition of DNA synthesis). If a compound orcombination of compounds or mixture of compounds inhibits DNA synthesis,this provides a basis for selecting and/or characterizing compounds fordevelopment and evaluation in indications benefiting from decreased cellproliferation (e.g., cancer including proliferating malignant cells andproliferating endothelial cells) and for evaluating efficacy, dosage,etc. if a compound or combination of compounds or mixture of compoundsstimulates DNA synthesis, then this provides a basis for selectingand/or characterizing compounds for development and evaluation intreating indications benefiting from increased cell proliferation (e.g.,wound healing) and for evaluating efficacy, dosage, etc.

As shown in FIG. 20, cell proliferation can be reduced byanti-proliferative agents, in this case gemcitabine and hydroxyurea.

Example 3: DNA Synthesis in Rat Hippocampal Neuroprogenitor Cells as aBiomarker of Neurogenesis

A compound or a combination of compounds or a mixture of compounds aretested on rats to determine whether they have effects on neurogenesis.Compounds with neurogenic potential (i.e., compounds that stimulateneurogenesis and/or inhibit neuroprogenitor cell death, includinginhibition of apoptosis and/or inhibition of necrosis) may find use intreating spinal cord injury, Parkinson's disease, Huntington's disease,and other neurodegenerative disorders. By detecting neurogenesis and orinhibition of neuroprogenitor cell death, the methods allow for theselecting and/or characterizing of compounds for developing andevaluating the agents for treating the disorders listed, supra.

Rats are divided into exposed and control groups and administeredlabeled water as in Example 1, supra. After exposure to a compound or acombination of compounds or a mixture of compounds or vehicle if controlrat, by gavage, intrathecal, or intracranial administration (route ofadministration is dependent on the chemistry of the compound,combination of compounds, or mixture of compounds, as is well known inthe art) rats are euthanized by CO₂ asphyxiation and whole brains areremoved.

For isolating tissue for neurogenesis analysis, the brain is bisectedlongitudinally and each hippocampal lobe is separated from theoverlaying cortical white matter using the natural separation line alongthe alveus hippocampus. The white matter of the fimbria and subiculumisis removed.

Tissues are finely minced and digested for 45 min in a solution ofpapain (2.5 U/ml; Worthington, Freehold, N.J.), DNase (250 U/ml,Worthington), and neutral protease (1 U/ml Dispase; Boehringer Mannheim,Indianapolis, Ind.) dissolved in HibernateA.

Whole digested tissue is then suspended in HibernateA, triturated with abarely fire-polished siliconized Pasteur pipet, and thoroughly mixedwith an equal volume of Percoll solution. The Percoll solution is madeby mixing nine parts of Percoll (Amersham Pharmacia Biotech, Uppsala,Sweden) with one part 10×PBS (Irvine Scientific, Santa Ana, Calif.).

The cell suspension is then fractionated by centrifugation for 30 min,18° C., at 20,000×g. Cell fractions are harvested and washed free ofPercoll by three or more rinses in HibernateA.

DNA synthesis is measured as in Example 2, supra. DNA is labeled by ²Has shown in FIGS. 2 and 3.

Exposed animals are then compared to unexposed animals to determine if acompound or a combination of compounds or a mixture of compoundsincreases DNA synthesis in hippocampal neuroprogenitor cells, whichprovides a basis for selecting and/or characterizing compounds fordevelopment and evaluation for treating neurodegenerative diseases andfor evaluating efficacy, dosages, etc.

Example 4: DNA Synthesis in Mouse Hippocampal Neuroprogenitor Cells as aBiomarker of Neurogenesis

Adult male mouse neuroprogenitor cell proliferation assays were preparedas described in Example 3, supra. Mice were chronically treated witheither vehicle, fluoxetine (10 mg/kg/day), or imipramine (20 mg/kg/day).Two weeks after initiation of drug treatment, mice were labeled with 10%²H₂O. Mice were sacrificed after 3 or 7 days of label, hippocampalprogenitor cells were isolated, followed by DNA isolation and GC_MSanalysis as described in Example 2, supra.

Chronic treatment with antidepressant drugs, imipramine and fluoxetine,produced a significant increase in the proliferation of mousehippocampal neuroprogenitor cells in the hippocampus (see FIGS. 7 and8). The magnitude of response is in accord with previous studies showingincreased cell proliferation by BrdU labeling following antidepressantdrug treatment.

Example 5: Measurement of Flux Rates of Aβ, APP, sAPP, and CTF in Miceas Biomarkers of Alzheimer's Disease

Alzheimer Disease (AD) is a progressive neurodegenerative disorder thatoccurs spontaneously with aging. There are multiple factors thatcontribute to AD, but one of the most important components is the Aβpeptide, which forms insoluble deposits in the brains of patients, andleads to a range of neurodegenerative events. The Aβ peptide is derivedfrom the beta-Amyloid Precursor Protein (APP), which is cleaved by a setof proteases to form Aβ. Perturbations in the processing of APP havebeen proposed to contribute to AD, as have changes in the rate ofclearance of Aβ from the CNS. Therapeutic interventions aimed ataltering the processing of APP, in order to reduce the generation of Aβ,provide a rational and widely pursued strategy for preventing ortreating AD. Drugs that alter the processing of APP and/or Aβ areexpected to make up the “next generation” of AD drugs.

Other drugs, including some in clinical trials, are focused on treatingor slowing the events downstream of Aβ generation and deposition. Suchdownstream processes include neuroinflammation, memory loss, neuronalcell death, and impairment of neurogenesis.

As stated above, Aβ, APP, secreted amyloid precursor protein (sAPP), andthe C-terminal fragment of APP (CTF) are important in the pathogenesisof AD. By measuring whether a compound, combination of compounds, ormixture of compounds can inhibit the synthesis of Aβ or APP or sAPP orCTF (or two or more of these proteins) one may in turn discover a novelmeans for treating AD.

The current benchmark for preclinical AD drug development is the APPtransgenic mouse model. In these mice, total Aβ deposits in the brainare enumerated histologically. A reduction in this “plaque load”measurement is an indicator of drug effect. The formation of plaques intransgenic mice takes months to years, however, and the mice areexpensive. A fast, quantitative preclinical assay of APP and/or Aβkinetics that can be used in normal mice to test the actions oftherapeutic interventions in the APP/Aβ pathway would greatly acceleratepre-clinical AD research. Isolation of Aβ or other peptides generated inthis pathway from urine, plasma or cerebrospinal fluid could allow thiskinetic approach to be applied in humans, as a biomarker of AD risk andresponse to therapies.

The flux of APP through the Aβ generating pathway will be a rapid andsensitive marker of efficacy for pre-clinical drug evaluation. Drugsthat downregulate APP production or block the processing of APP by theAβ generating protease β-secretase can be identified, compared foractivity, and optimized through APP kinetics. An advantage of thisapproach is that these measurements can be made in young, wild-typeanimals, as well as in transgenic models of AD. A further advantage ofthis approach is that it is expected to be significantly faster (weeksto months vs. months to years) than waiting for transgenic animals todevelop plaques.

Mice are labeled with ²H₂O using the procedures described in Example 1,supra, for rats. Mice are given a compound, or a combination ofcompounds, or a mixture of compounds via gavage, intrathecal, orintracranial administration. Urine is collected to isolate Aβ protein.Total urinary protein is concentrated and exchanged in a suitable bufferfor immunoaffinity purification. After immunoaffinity purification, Aβcan be further purified using size exclusion and/or reversed phasechromatography. The identity of purified peptides is confirmed by ELISA,western blot, and LC-MS (ESI).

Alternatively, mice are sacrificed and brain tissue is extracted and APPand CTF are obtained. Secreted APP is extracted from mouse cerebralspinal fluid (CSF) or brain. Proteins are extracted in neutral buffer,insoluble material is removed, and proteins precipitated. Resultingmaterial is exchanged into an ion exchange buffer, and purified by ionexchange chromatography and then size exclusion and/or reversed phasechromatography. The identity of purified protein is confirmed by ELISAand western blot.

Purified proteins are hydrolyzed by treating with 6 N HCl, 16 hours at110° C. Hydrolysates are dried and the N,O-penatfluorobenzyl derivativeis generated by addition of PFBBr (Pierce) at 100° C. for 1 hour.Derivatized hydrolysates are extracted with ethyl acetate, dried withNaSO₄, and analyzed on a DB225 GC column, starting temp 100° C.increasing 10° C./min to 220° C. Alanine is analyzed with selected ionmonitoring of m/z 448,449; other amino acids including glycine,methionine, leucine, isoleucine, and tyrosine also can be analyzed.

Enrichments for Aβ, APP, and CTF are performed as described, supra.Molecular flux rates for Aβ, APP, and CTF are calculated as described,supra. Exposed animals are then compared to unexposed animals todetermine if a compound, or a combination of compounds, or a mixture ofcompounds inhibits Aβ, APP, sAPP, and CTF synthesis and/or stimulatestheir degradation, which provides a basis for selecting and/orcharacterizing compounds for development and evaluation for treating ADand for evaluating efficacy, dosages, etc.

Example 6: Glycolytic Disposal of Glucose in Normal Rats as a Biomarkerfor Insulin Resistance, Type II Diabetes, Metabolic Syndrome, andCardiovascular Disease

Glycolytic disposal of a glucose load reflects several insulin sensitivemetabolic steps including uptake, phosphorylation, and glycolyticmetabolism of blood glucose. Accordingly, whole body glycolytic rate isa biomarker for insulin resistance, metabolic syndrome, cardiovasculardisease, and type II diabetes (see Reaven G M. Banting Lecture 1988.Role of insulin resistance in human disease. Diabetes 37(12):1595-607,1988). Rats, as in Example 1, supra, are used to measure glycolyticdisposal in vivo in response to a compound, or a combination ofcompounds, or a mixture of compounds for effects on insulin sensitivity.Because insulin resistance (lack of insulin sensitivity) underliesnumerous diseases of Western society (Reaven), measurement of glycolyticdisposal finds use in identifying and characterizing compounds fordeveloping and evaluating therapeutics for insulin resistance, metabolicsyndrome, cardiovascular disease, and type II diabetes.

More specifically, the method may be used to determine newly synthesizedglycogen. Newly synthesized glycogen synthesis can be determinedindirectly by subtracting glycolysis from the total amount of glucoseinitially administered since the total disappearance of glucose is equalto the total amount of glycolysis+the total amount of newly synthesizedglycogen. The following equation can be used to calculate newlysynthesized glycogen:Total glucose utilization−glycolysis=newly synthesized glycogen

The ²H-glucose labeling protocol consists of an initial intraperitoneal(ip) injection or oral administration of 99.9% [6,6-²H₂] glucose. Forlabeling rats, 2 mg labeled glucose per gram body weight is introduced.Body water is collected as serum at various timepoints. A compound or acombination of compounds or a mixture of compounds is administered by anappropriate route of administration such as gavage.

Glycolysis is measured by measuring deuterium in body water as a percentof administered [6,6-²H₂] glucose normalized to account for differentmolar quantities of deuterium in molecular glucose and molecular water.Deuterated water is measured as described, supra. Glycolysis fromexposed rats is compared with glycolysis from unexposed rats todetermine if a compound or a combination of compounds or a mixture ofcompounds increased glycolysis, which provides the basis for selectingand characterizing compounds for development and evaluation for treatinginsulin resistance, type H diabetes, and/or other metabolic disordersand for evaluating efficacy, dosages, etc.

As shown in FIG. 24, rosiglitazone, a known insulin sensitizing agent,can improve insulin sensitivity in Zucker-Diabetes-Fat (ZDF) rats, ananimal model of insulin resistance and pre-diabetes.

FIG. 29 shows a decrease in glucose utilization as measured as a percentof total deuterated glucose administered. As can be seen, the rats fed ahigh fat diet for three weeks had an impaired ability to metabolizeglucose compared to control rats who were fed a normal diet (results arestatistically significant with p<0.05).

FIG. 30 shows glucose utilization in a number of human subjects groupedinto lean, overweight, obese, type II diabetes, and HIV-infectedindividuals. All of the groups showed impaired glucose utilization withrespect to the lean group, which is consistent with established dataindicating overweight, obese, and HIV+ individuals are more likely to beinsulin resistant. As expected, type II diabetes subjects were shown tobe insulin resistant.

Example 7: Brain Galactocerebroside Turnover as a Biomarker ofRemyelination in Demyelinating Diseases

Brain galactocerebroside turnover is a fundamental biomarker fordemyelination, the underlying biochemical process (metabolic pathway) indemyelination diseases such as multiple sclerosis (MS). Rats are givendeuterated water as in Example 1, supra. Rats are administered acompound, or a combination of compounds, or a mixture of compounds viagavage, intrathecal, intraperitoneal, or intracranial administration. Ifit is found that a compound, or a combination of compounds, or a mixtureof compounds stimulates remyelination and/or inhibits demyelination,this serves as a basis for selecting and characterizing compounds fordevelopment and evaluation for treating MS and other demyelinatingdiseases.

Weigh a set of 2-mL microcentrifuge tubes. Brains are collected from rator mouse carcasses and weighed. The brain is put onto an ice-cooledglass plate, and 10 crystals of BHT are added. A razor blade is used tomince the brain for 1 minute. A spatula is used to put the minced brainback into the microcentrifuge tubes. The brain is minced well with aspatula. 80-120 mg of minced brain is put into 13×100 mm glass tubeswith PTFE screw caps ensuring the tissue is at the bottom of the tube.The rest of the brain is stored in the microcentrifuge tubes at −20° C.2 mL of chloroform-methanol 2:1 (v/v) with BHT is added into the glasstubes and the tubes are vortexed ensuring that all of the tissues aresoaked in the solvent. Stand at least 5 minutes at room temperature. Thetubes are centrifuged at 2000 RCF for 10 minutes at room temperature.The supernatant (lipid extracts) is poured into 2-mL screw capped vialsand the solid residue is discarded.

TLC is performed. A 20 mL pipette is used to spot 20 mL of totalcerebroside standard on lanes 1, 10, 19 of Whatman LK6DF silica gel 60TLC plates. For each sample 40 μL of lipid extracts are spotted on eachlane The TLC plates are developed using 69.2% chloroform, 26.6%methanol, 4.2% water developing solvent. After TLC plates develop, wait15 minutes for the plates to dry. 20 iodine crystals are put into a tankspecially used for iodine vapor. The tank is put on a heatblock set at80° C. The dried TLC plates are put in the iodine tank to visualize thespots of lipids containing double bonds. The spots of total cerebrosidestandard are matched with those of samples. The silica gel is collectedonto weighing paper and transferred to a 12×75 mm disposable glass tube.2 ml of 65% dichloromethane/25% methanol/10% ammonium hydroxide solutionis added and vortexed. Let stand until silica settles. The solvent ispoured into a 13×100 mm screw cap tube and dried down. The sample isthen resuspended in 1 ml of chloroform-methanol 2:1 with BHT and 1 mL of3N methanolic HCl is added into the tube and the tube is capped tightly.The tubes are put on a heatblock at 80° C. for 1 h. The tubes are thenremoved from the heating block and allowed to cool to room temperature.1.5 mL H₂O and 3 mL hexane are added into the tubes and the tubes arevortexed. 1.8-2 mL of the bottom layer (methyl glucose and methylgalactose) are transferred to GC vials. The GC vials dried in thespeedvac. Following drying 100 μl of freshly made aceticanhydride-pyridine 2:1 (v/v) is added to the GC vials and the vials arecovered and allowed to stand for 1 h at room temperature. The vials arethen blown down under N₂ until dry. 100 μL ethyl acetate is. The samplesare run on the GC/MS and galactocerebroside enrichments are determined.The molecular flux rates of galactocerebroside is determined asdescribed supra, from rats exposed to a compound or a combination ofcompounds or a mixture of compounds and unexposed (vehicle control)rats. Enrichments of galactocerebroside greater than galactocerebrosideenrichments in control animals indicates increases synthesis ofgalactocerebroside and possible remyelination (which, as discussedsupra, provides a basis for selecting and/or characterizing a compoundfor development and evaluation for treating MS and other demyelinatingdiseases and for evaluating efficacy, dosages, etc.).

Enrichments that are less than controls indicates reduced myelinsynthesis (pointing to neuronal toxicity, specifically toxicity to themyelin sheath; this will find use in identifying neurotoxic chemicalssuch as new industrial solvents).

As shown in FIG. 26, cuprizone, a known demyelinating toxic agent, isshown to suppress synthesis of galactocerebroside (GalCer) in the brain.After removal of cuprizone, fractional synthesis of GalCer is increasedabove the normal rate during the remyelinating phase.

Example 8: Collagen Turnover in Rats as a Biomarker of Osteoarthritis

The loss of cartilage from the articular surface is a principal featurein advanced osteoarthritis (OA). Considerable evidence supports thehypothesis that this loss is due, at least in part, to increaseddegradation rates of collagen. Thus, collagen turnover (degradation ofcollagen into its constituent breakdown products, which are principallyhydroxyproline and telopeptides) is a fundamental biomarker in theunderlying biochemical process (metabolic pathway) of OA and other jointdiseases involving cartilage destruction (for a depiction of collagenlabeling see FIG. 4; for its biosynthetic and degradative pathways, seeFIG. 5).

Normal female rats (13 weeks of age, Sprague Dawley) will be labeledwith ²H₂O by the following protocol: at time 0 an ip injection ofsterile 100% ²H₂O 0.9% NaCl will be administered (30 ml/kg). Drinkingwater will be replaced with a solution containing 8% ²H₂O and maintaineduntil sacrifice.

Rats are administered a compound or a combination of compounds or amixture of compounds via an appropriate route of administration. If itis found that a compound or a combination of compounds or a mixture ofcompounds inhibits collagen degradation (by, for example decreasingenrichment of degradation products of collagen including peptides, freehydroxyproline and/or telopeptides), this serves as a basis forselecting and/or characterizing compounds for development and evaluationfor treating OA and other joint diseases involving cartilage destructionand for evaluating efficacy, dosages, etc.

Rats (five per time point) will be collected after 2, 4, 8, and 12 daysof ²H₂O labeling. Articular cartilage will be collected from hindlimbknee surfaces (MTP, LTP, MFC, LFC) and the femoral head and placed intopre-weighed RNAse free tubes, snap frozen and stored at −70 C untilanalyses.

Collagen is isolated by initially homogenizing tissue in 0.1 M NaOH.Collagen is purified from as little as 10 mg of fresh or frozen totalliver homogenate as follows: using a Polytron homogenizer, collagen isisolated from soft tissue by homogenizing in 0.5 mL 100 mM NaOH. Underthese conditions, collagen remains insoluble while most other proteinsare readily dissolved. After centrifugation at 7,000×g for 10 minutes at4° C., the supernatant is discarded. The pellet is washed briefly with 2mL H₂O and solubilized in reducing Laemmli sample buffer (Bio-Rad,Hercules, Calif.) after boiling for 3 minutes. The dissolved material issize-fractionated by SDS-PAGE. Using standard techniques, proteins aresubsequently transferred onto PVDF, and a collagen band corresponding tothe alpha monomer of collagen is excised from the resulting membraneafter staining the membrane with Coomassie blue.

Degradative products of collagen (e.g., free hydroxyproline,collagen-derived peptides, telopeptides) are initially concentrated byreversed phase solid phase extraction. This is followed by a series ofchromatographic steps which include size exclusion, anion exchange, andreversed phase separation. During the isolation protocol, the presenceof specific degradation products is monitored both by commerciallyavailable immunological reagents Cartilaps (Nordic Bioscience, Herlew,Denmark) as well as by electrospray ionization mass spectrometry(ES/MS). For ES/MS, a selected ion monitoring is based on masses of themost commonly occurring variant of CTxII, with molecular weights of1592.64 being used. The turnover of collagen measured in the articularcartilage will be compared between each site collected and to the CTxIIpeptides isolated from the synovial fluid.

Degradative products of collagen (e.g., free hydroxyproline,collagen-derived peptides, telopeptides) are hydrolyzed by treating with6 N HCl, 16 hours at 110° C. Hydrolysates are dried and theN,O-penatfluorobenzyl derivative is generated by addition of PFBBr(Pierce) at 100° C. for 1 hour. The hydroxyl group of hydroxyproline isfurther derivatized with methyl imidazole/MTBSTFA. Hydroxyproline isanalyzed on a DB225 GC column, starting temp 100° C. increasing 10°C./min to 220° C. with selected ion monitoring of m/z 424,425.

Synthesis rates will be measured and calculated as described, supra.

Example 9: Liver Cell Turnover as a Biomarker for Subclinical LiverToxicity or Disease

Liver cell turnover is a biomarker for liver injury and disease. Infact, liver cell proliferation (in response to exposure to environmentalcontaminants or therapeutic compounds or other factors such as hepatitisviruses) occurs well before clinical manifestations of injury ordisease. Measuring liver cell proliferation in vivo, in response toexposure to a compound, or a combination of compounds, or a mixture ofcompounds, for example, exposure to a toxic chemical (e.g., a newindustrial chemical, an environmental pollutant, or a known livertoxicant such as carbon tetrachloride) or an environmental toxin (e.g.,biological factor eliciting a toxic effect) or exposure to a chemicalentity (whether new or old), or a drug candidate, or a drug lead, or analready-approved drug, or a biological factor is a sensitive method,therefore, for detecting liver changes before clinical injury or diseaseoccurs. The deoxyribose (dR) moiety of dNTPs in replicating DNA can belabeled endogenously, through the de novo nucleotide synthesis pathway,using stable isotope-labeled glucose or ²H₂O (FIG. 1). In this example,rats are labeled with ²H₂O using the procedures described in Example 1,supra. Control and exposed groups are used as described in Example 1,supra.

Mice are given CCl₄ i.p. twice weekly for up to 4 weeks, and continuousoral ²H₂O is given throughout, following an i.p. bolus of ²H₂O. Liversare perfused in situ with saline to minimize blood cell contamination.Livers are homogenized and DNA from 5-mg aliquots is analyzed for ²Hincorporation by gas chromatography/mass spectrometry analysis, afterisolation and hydrolysis of genomic DNA, as described in Example 1,supra. Livers are collected 4 days after the last dose of CCl₄. The cellproliferation rate can then be calculated from the enrichment in the DNAof the target cell compared to measured body water enrichment or to areference cell type which is fully replaced.

DNA is extracted from a liver sample, either by biopsy or liverhomogenate. Liver cell proliferation can be determined on a sample asmall as 2 mg (400,000 cells). The use of total liver, rather thanisolated hepatocytes, allows for efficient scale-up and the sampleprocessing can be adapted to a 96-well automated system for extremelyhigh throughput. Total liver cell proliferation will be compared to themeasured proliferation of isolated hepatocytes, and non-parenchymalcells for validation. Total liver cell proliferation is a simpler methodto employ than isolated hepatocyte proliferation, therefore if it isfound that measuring total liver cell proliferation approximatesisolated hepatocyte proliferation, then using total liver cellproliferation will be preferable to using isolated hepatocyteproliferation in detecting subclinical liver toxicity.

FIG. 13 shows the effects of CCl₄ on liver cell proliferation over 7days of treatment. Swiss Webster mice were given IP injections of CCl₄over 7 days concurrent with ²H₂O. Total liver cell turnover (i.e.,increased proliferation of liver cells) was evident with increasingdoses of CCl₄ reflecting the liver's response to the toxic insult.

Example 10: Collagen Synthesis as a Biomarker of Liver Fibrosis

Fibrosis, which is an overproduction of extracellular matrix (ECM)components, is a hallmark of many diseases of the vascular system,heart, lung, liver, and kidneys, and skin (for a depiction of collagenlabeling see FIG. 4; for its biosynthetic and degradative pathways, seeFIG. 5). Fibrosis generally occurs in response to tissue injury fromtoxicants such as alcohol as well as from mechanical and oxidativestresses. The most notable feature of tissue fibrosis is the chronicenhancement of biosynthesis and reduced degradation of collagen;eventually this buildup reduces organ function and leads to organfailure.

Early, predictive diagnosis of tissue fibrosis is critical for theassessment of drug toxicity and disease treatment. However, existingbiomarkers of fibrosis are often expensive and insensitive. Theseinclude endpoint assays for measurement of increased collagen pool sizeand histochemical staining of ECM components within tissue biopsies.Measurements of alterations in collagen synthesis are more sensitive andquantitative than measurements of pool size or qualitativehistopathology scoring.

Hepatic fibrosis, the accumulation of excessive extracellular matrix(collagen), is a common result of chronic liver injury or disease.Chronic, untreated fibrosis advances to cirrhosis, which isirreversible. Often associated with chronic alcohol abuse, fibrosis canresult from other drug toxicities (including adverse effects fromchemotherapeutics such as fenofibrate, griseofulvin, or methotrexate)and from exposure to environmental chemicals (e.g., new industrialchemicals or known industrial chemicals such as carbon tetrachloride).In fact, any drug that causes low persistent hepatic damage could leadto fibrosis which may not appear in clinical practice until many yearsafter wide spread use.

In this example, rats are labeled with ²H₂O using the proceduresdescribed in Example 1, supra. Control and exposed groups are used asdescribed in Example 1, supra. Exposed rats are given a single dose ofdiethylnitroseamine (200 mg/kg). Diethylnitroseamine is a potenthepatotoxin, carcinogen and mutagen and has been shown to inducefibrosis from a single dose. Rats will receive ²H₂O continuously for upto 2 weeks, then animals will be sacrificed at 7 and 14 days posttreatment.

Collagen is purified from as little as 10 mg of fresh or frozen totalliver homogenate as follows: using a Polytron homogenizer, collagen isisolated from soft tissue by homogenizing in 0.5 mL 100 mM NaOH. Underthese conditions, collagen remains insoluble while most other proteinsare readily dissolved. After centrifugation at 7,000×g for 10 minutes at4° C., the supernatant is discarded. The pellet is washed briefly with 2mL H₂O and solubilized in reducing Laemmli sample buffer (Bio-Rad,Hercules, Calif.) after boiling for 3 minutes. The dissolved material issize-fractionated by SDS-PAGE. Using standard techniques, proteins aresubsequently transferred onto PVDF, and a collagen band corresponding tothe alpha monomer of collagen is excised from the resulting membraneafter staining the membrane with Coomassie blue.

Collagen degradative products are derivatized and analyzed as describedin Example 8, supra.

Hydroxyproline is a molecule of interest and is measured as OH-proline,the molecule being essentially unique to collagen. Because of this fact,total liver protein hydrolysate can be derivatized and the ²H enrichmentof hydroxyproline determined by GC/MS. Fractional synthesis of collagenin normal and diethylnitroseamine-treated animals is calculated from ²Hincorporation into hydroxyproline from total liver protein. In thisfashion collagen synthesis can be determined with a minimal amount ofsample preparation, lending itself to high-throughput analysis.Therefore, any entity (or combinations of entities) can be screened todetermine whether collagen synthesis occurs in response to exposure. Ifcollagen synthesis is observed, the organism is at increased risk forliver fibrosis. The method therefore allows for the screening of acompound, or a combination of compounds, or a mixture of compounds todetermine whether they induce collagen synthesis in the liver andtherefore possess hepatotoxic effects that place the exposed organism atincreased risk for liver fibrosis and cirrhosis.

Conversely, if it is found that a compound, or a combination ofcompounds, or a mixture of compounds inhibits or reduces collagensynthesis and/or enhances collagen degradation, then this provides thebasis for selecting and/or characterizing the compound for developmentand evaluation for treating liver fibrosis, and for evaluating efficacy,dosages, etc.

As shown in FIG. 19, CCl₄, a known fibrotic agent, increases collagensynthesis in mouse liver, an effect that is suppressed by twoanti-fibrotic agents (a) interferon-gamma and (b) rosiglitazone.

Example 11: Collagen Synthesis as a Biomarker of Pulmonary Fibrosis

Fibrosis, which is an overproduction of extracellular matrix (ECM)components, is a hallmark of many diseases of the vascular system,heart, lung, liver, and kidneys, and skin (for a depiction of collagenlabeling see FIG. 4; for its biosynthetic and degradative pathways, seeFIG. 5). Fibrosis generally occurs in response to tissue injury fromtoxicants such as alcohol as well as from mechanical and oxidativestresses. The most notable feature of tissue fibrosis is the chronicenhancement of biosynthesis and reduced degradation of collagen;eventually this buildup reduces organ function and leads to organfailure.

Early, predictive diagnosis of tissue fibrosis is critical for theassessment of drug toxicity and disease treatment. However, existingbiomarkers of fibrosis are often expensive and insensitive. Theseinclude endpoint assays for measurement of increased collagen pool sizeand histochemical staining of ECM components within tissue biopsies.Measurements of alterations in collagen synthesis are more sensitive andquantitative than measurements of pool size or qualitativehistopathology scoring.

Pulmonary fibrosis may arise from exposure to a broad spectrum ofairborne chemical pollutants and particulates, from sarcoidosis, as wellas exposure to certain pharmacological agents such as carmustine.Idiopathic forms of pulmonary fibrosis in which etiology is unclear alsoexist.

Normal male rats (6 to 9 weeks of age, Sprague Dawley) are labeled with²H₂O by the following protocol: at time 0 an ip injection of sterile100% ²H₂O 0.9% NaCl is administered (30 mL/Kg). Drinking water is thenreplaced with a solution containing 8% ²H₂O which is maintained untilsacrifice.

Rats are administered a compound, or a combination of compounds, or amixture of compounds via an appropriate route of administration such asip injection. If it is found that a compound, or a combination ofcompounds, or a mixture of compounds inhibits or reduces collagensynthesis and/or enhances collagen degradation, this provides a basisfor selecting and/or characterizing compounds for development andevaluation or treating pulmonary fibrosis and for evaluating efficacy,dosages, etc.

Conversely, if it is found that a compound, or a combination ofcompounds, or a mixture of compounds augments collagen synthesis and/orreduces collagen degradation, this provides a basis for reportingpotential, hitherto unpublished toxicities of new chemical entities,drug candidates, drug leads, already-approved drugs, biological factors,environmental chemicals, new lead compounds and the like.

Rats (five per time point) are euthanized after 2, 7, 14, 21, and 28days of ²H₂O labeling. Tissues including but not limited to skin, lung,liver, heart, and kidney will be removed and stored at −20° C. untilanalyses.

Collagen is preferentially precipitated by initially homogenizing tissuein 0.1 M NaOH. The homogenate is centrifuged at 7,000×g, and theresulting pellet is size-fractionated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Proteins are transferred to PVDFsolid support. Collagen is excised from the blot and hydrolyzed at 110°C. for 16 hours. Amino acids are derivatized and the ²H content intreatment groups is measured and analyzed as a function of time,relative to control groups.

Collagen degradative products are derivatized and analyzed as describedin Example 8, supra.

Example 12: Collagen Synthesis as a Biomarker of Myocardial Fibrosis

Fibrosis, which is an overproduction of extracellular matrix (ECM)components, is a hallmark of many diseases of the vascular system,heart, lung, liver, and kidneys, and skin (for a depiction of collagenlabeling see FIG. 4; for its biosynthetic and degradative pathways, seeFIG. 5). Fibrosis generally occurs in response to tissue injury fromtoxicants such as alcohol as well as from mechanical and oxidativestresses. The most notable feature of tissue fibrosis is the chronicenhancement of biosynthesis and reduced degradation of collagen;eventually this buildup reduces organ function and leads to organfailure.

Early, predictive diagnosis of tissue fibrosis is critical for theassessment of drug toxicity and disease treatment. However, existingbiomarkers of fibrosis are often expensive and insensitive. Theseinclude endpoint assays for measurement of increased collagen pool sizeand histochemical staining of ECM components within tissue biopsies.Measurements of alterations in collagen synthesis are more sensitive andquantitative than measurements of pool size or qualitativehistopathology scoring.

Myocardial fibrosis is a key feature of diseases of the heart. The mostcommon cause is coronary arteriosclerosis. Other causes include: 1,relative coronary insufficiency due to cardiac hypertrophy due tohypertension, valvular disease; 2, healed rheumatic myocarditis; 3,healed infectious, immune, toxic, or idiopathic myocarditis; 4,scleroderma.

Normal male rats (6 to 9 weeks of age, Sprague Dawley) are labeled with²H₂O by the following protocol: at time 0 an ip injection of sterile100% ²H₂O 0.9% NaCl is administered (30 mL/Kg). Drinking water is thenreplaced with a solution containing 8% ²H₂O which is maintained untilsacrifice.

Rats are administered a compound, a combination of compounds, or amixture of compounds via an appropriate route of administration such asip injection. If it is found that a compound, or a combination ofcompounds, or a mixture of compounds reduces collagen synthesis and/orenhances collagen degradation in myocardial tissue, this provides abasis for selecting and/or characterizing compounds for development andevaluation for treating myocardial fibrosis and for evaluating efficacy,dosages, etc.

Contrastingly, if it is found that a compound, or a combination ofcompounds, or a mixture of compounds augments collagen synthesis and/orreduces collagen degradation, this provides a basis for reportingpotential, hitherto unpublished myocardial toxicities of thosecompounds, combinations of compounds, or mixtures of compounds.

Rats (five per time point) are euthanized after 2, 7, 14, 21, and 28days of ²H₂O labeling. Tissues including but not limited to skin, lung,liver, heart, and kidney will be removed and stored at −20° C. untilanalyses.

Collagen is preferentially precipitated by initially homogenizing tissuein 0.1 M NaOH. The homogenate is centrifuged at 7,000×g, and theresulting pellet is size-fractionated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Proteins are transferred to PVDFsolid support. Collagen is excised from the blot and hydrolyzed at 110°C. for 16 hours. Amino acids are derivatized and the ²H content intreatment groups is measured and analyzed as a function of time,relative to control groups.

Collagen degradative products are derivatized and analyzed as describedin Example 8, supra.

Example 13: Collagen Synthesis as a Biomarker of Dermal Fibrosis

Fibrosis, which is an overproduction of extracellular matrix (ECM)components, is a hallmark of many diseases of the vascular system,heart, lung, liver, kidneys, and skin (for a depiction of collagenlabeling see FIG. 4; for its biosynthetic and degradative pathways, seeFIG. 5). Fibrosis generally occurs in response to tissue injury fromtoxicants such as alcohol as well as from mechanical and oxidativestresses. The most notable feature of tissue fibrosis is the chronicenhancement of biosynthesis and reduced degradation of collagen;eventually this buildup reduces organ function and leads to organfailure.

Early, predictive diagnosis of tissue fibrosis is critical for theassessment of drug toxicity and disease treatment. However, existingbiomarkers of fibrosis are often expensive and insensitive. Theseinclude endpoint assays for measurement of increased collagen pool sizeand histochemical staining of ECM components within tissue biopsies.Measurements of alterations in collagen synthesis are more sensitive,responsive, and quantitative than measurements of pool size orqualitative histopathology scoring.

Dermal fibrosis arises as a part of the pathology of scleroderma,sclerodermoid disorders, graft versus host disease, severe acne andother disorders. Halofuginone and other antifibrotic agents are beinginvestigated and prescribed for such treatment, but this field wouldgreatly accelerate with the advent of better tests for the earlydetection of such diseases and their animal models.

Normal male rats (6 to 9 weeks of age, Sprague Dawley) are labeled with²H₂O by the following protocol: at time 0 an ip injection of sterile100% ²H₂O 0.9% NaCl is administered (30 mL/Kg). Drinking water is thenreplaced with a solution containing 8% ²H₂O which is maintained untilsacrifice.

Rats are administered a compound, a combination of compounds, or amixture of compounds via an appropriate route of administration such asip injection. If it is found that a compound, or a combination ofcompounds, or a mixture of compounds reduces collagen synthesis and/orenhances collagen degradation, this provides a basis for selectingand/or characterizing compounds for development and evaluation fortreating dermal fibrosis and for evaluating efficacy, dosages, etc.

Conversely, if it is found that a compound, or a combination ofcompounds, or a mixture of compounds augments collagen synthesis and/orreduces collagen degradation, this provides a basis for reportingpotential, hitherto unpublished toxicities of those compounds,combinations of compounds, or mixtures of compounds.

Rats (five per time point) are euthanized after 2, 7, 14, 21, and 28days of ²H₂O labeling. Tissues including but not limited to skin, lung,liver, heart, and kidney will be removed and stored at −20° C. untilanalyses.

Collagen is preferentially precipitated by initially homogenizing tissuein 0.1 M NaOH. The homogenate is centrifuged at 7,000×g, and theresulting pellet is size-fractionated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Proteins are transferred to PVDFsolid support. Collagen is excised from the blot and hydrolyzed at 110°C. for 16 hours. Amino acids are derivatized and the ²H content intreatment groups is measured and analyzed as a function of time,relative to control groups.

Collagen degradative products are derivatized and analyzed as describedin Example 8, supra.

Example 14: Collagen Synthesis as a Biomarker of Renal Fibrosis

Fibrosis, which is an overproduction of extracellular matrix (ECM)components, is a hallmark of many diseases of the vascular system,heart, lung, liver, and kidneys (for a depiction of collagen labelingsee FIG. 4; for its biosynthetic and degradative pathways, see FIG. 5).Fibrosis generally occurs in response to tissue injury from toxicantssuch as alcohol as well as from mechanical and oxidative stresses. Themost notable feature of tissue fibrosis is the chronic enhancement ofbiosynthesis and reduced degradation of collagen; eventually thisbuildup reduces organ function and leads to organ failure.

Early, predictive diagnosis of tissue fibrosis is critical for theassessment of drug toxicity and disease treatment. However, existingbiomarkers of fibrosis are often expensive and insensitive. Theseinclude endpoint assays for measurement of increased collagen pool sizeand histochemical staining of ECM components within tissue biopsies.Measurements of alterations in collagen synthesis are more sensitive andquantitative than measurements of pool size or qualitativehistopathology scoring.

In the kidney, fibrosis is characterized by long, gradual replacement ofhealthy tissue with fibrotic tissue. Unlike typical wound healingresponses, the kidney, when subjected to toxic insult or similarlesion-generating event, continues to produce extracellular matrixproteins including collagen long after the initial event. Antifibroticagents for the treatment of renal fibrosis are the subject of intenseresearch.

Normal male rats (6 to 9 weeks of age, Sprague Dawley) are labeled with²H₂O by the following protocol: at time 0 an ip injection of sterile100% ²H₂O 0.9% NaCl is administered (30 mL/Kg). Drinking water is thenreplaced with a solution containing 8% ²H₂O which is maintained untilsacrifice.

Rats are administered a compound, a combination of compounds, or amixture of compounds via an appropriate route of administration such asip injection. If it is found that a compound, a combination ofcompounds, or a mixture of compounds reduces collagen synthesis and/orenhances collagen degradation, this provides a basis for selectingand/or characterizing compounds for development and evaluation fortreating renal fibrosis and for evaluating efficacy, dosages, etc.

Conversely, if it is found that a compound, or a combination ofcompounds, or a mixture of compounds augments collagen synthesis and/orreduces collagen degradation, this provides a basis for reportingpotential, hitherto unpublished toxicities of compounds, combinations ofcompounds, or mixtures of compounds.

Rats (five per time point) are euthanized after 2, 7, 14, 21, and 28days of ²H₂O labeling. Tissues including but not limited to skin, lung,liver, heart, and kidney will be removed and stored at −20° C. untilanalyses.

Collagen is preferentially precipitated by initially homogenizing tissuein 0.1 M NaOH. The homogenate is centrifuged at 7,000×g, and theresulting pellet is size-fractionated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. Proteins are transferred to PVDFsolid support. Collagen is excised from the blot and hydrolyzed at 110°C. for 16 hours. Amino acids are derivatized and the ²H content intreatment groups is measured and analyzed as a function of time,relative to control groups.

Collagen degradative products are derivatized and analyzed as describedin Example 8, supra.

Example 15: Neurogenesis as a Biomarker of Neurotoxicity andNeurodegeneration

Tissue from the cerebral cortex is isolated by dissection from freshlykilled, ²H₂O-labeled mice. Total cerebral cortical DNA is isolated fromapproximately 25 mg of tissue using a commercially available kit(Qiagen, Valencia, Calif.), and then hydrolyzed and derivatized forGC/MS as described in Example 2, supra. Deuterium incorporation isdetermined by GC/MS and used to determine cell proliferation rates inthe cerebral cortex. These rates can reflect a variety of processes,most notably gliogenesis (as part of brain development, as a response toinjury, or as a response to an administered agent). Such rates may alsoreflect neuroinflammation, which can stimulate microglial cellproliferation. Neurotoxicity, which can cause neurodegeneration andsubsequent gliogenesis or neuroinflammation, may also have effects onthe cerebral cortex which can be detected by the above method.Neurogenesis, which occurs more slowly in the cerebral cortex, will alsobe detectable by the same method. These rates may also reflect theinfiltration of other cell types, such as macrophages, into the cerebralcortex. This technique can be adapted to study cell proliferation in anysubstructure of the brain that can be isolated by dissection, and willyield the same type of information for that tissue. A compound, or acombination of compounds, or a mixture of compounds can therefore betested for anti-inflammatory, microglial cell proliferation,neurogenesis, and/or gliogenesis activity. A compound, combination ofcompounds, or a mixture of compounds having such activity is a candidatefor development and evaluation for treating brain injury and/or braindisease.

Conversely, a compound, or a combination of compounds, or a mixture ofcompounds can be screened for neurotoxicity. FIG. 12 shows one suchtoxin, lipopolysaccharide (LPS), in which the high dose administeredcaused increased cortical cell turnover in response to the neurotoxiceffects of LPS.

Example 16: Pancreatic β Cell Turnover as a Biomarker for Diabetes

The number of functionally intact pancreatic β cells in the islet ofLangerhans is of decisive importance for the development, course, andoutcome of diabetes mellitus. Generally speaking, the total β-cell massreflects the balance between the renewal and loss of these cells.Virtually all forms of diabetes mellitus are characterized by aninsufficient extent of β cell replication needed to compensate for theloss or dysfunction of β cells occurring in diabetes. A reduction of theβ-cell mass in the pancreas is, in fact, the critical clinical event inthe development of type 1 diabetes and a reduced islet mass incombination with insulin resistance is necessary for type 2 diabetes todevelop. Type 1 diabetes develops as a result of autoimmune destructionof the β cells; type 2 diabetes is characterized by development of earlyinsulin resistance and a failure of the β cells to compensate for thehyperinsulinemia. Insufficient production of biologically active insulinis a common denominator in almost all forms of diabetes and the degreeof insulin deficiency determines both the severity of the disease andthe choice of therapy. Therefore, it is imperative to develop methods ofmeasuring islet proliferation, as islet proliferation is a surrogate forpancreatic β-cell regeneration. A 50% partial pancreatectomy of rats canbe performed to mimic the pre-diabetic state, especially in order tostudy β-cell regeneration (for reviews see Risbud M. V and Bhonde R. R.Diabetes Res Clin Pract. 2002 December; 58(3):155-65; Kulkarni R. N. IntJ Biochem Cell Biol. 2004 March; 36(3):365-71).

Pancreatectomized (50%) male Wistar rats, and weight and age-matchcontrols are obtained from commercial sources (Charles River,Wilmington, Mass.). Rats are given free access to water and standardchow. The animals are housed in a temperature-controlled room with a12-h light, 12-dark cycle. Rats are treated with drugs or vehicles asdetermined by the study requirements.

Rats are killed by CO₂ gas, and the pancreatic duct is identified andcannulated for intraductal collagenase injection. 30 mL type Vcollagenase solution (20 mg/30 mL; Sigma Chemical Co. St Louis, Mo.)diluted in Hanks' Balanced Salt Solution (HBSS) buffer is injected intothe pancreatic duct after cannulation. The pancreas is inflated,carefully removed, and placed in a 25-mL flask with 5 mL coldcollagenase. The pancreas is digested in water bath at 37° C. for 15min. At the end of the digestion, the pancreatic digest is washed withfresh HBSS. The islets are purified by Ficoll gradient. Approximately200-250 intact islets are obtained per pancreas.

DNA is isolated from the islets and isotopic enrichment is measured asdescribed in Example 2, supra.

A compound, or a combination of compounds, or a mixture of compounds cantherefore be tested for the ability to stimulate islet proliferation orinhibit islet degradation. The islet of Langerhans serves as a surrogatemarker for pancreatic β cell proliferation. A compound having suchactivity is a candidate for development and evaluation as an agent fortreating diabetes.

Data obtained using the methods described herein is shown in FIG. 9.Control or pancreatectomized animals were administered ²H₂O for 14 days,and Islet (beta-cell) proliferation was measured. Pancreatectomizedanimals showed increased proliferation after 14 days of ²H₂O(19.31%±4.0, n=3, vs. 11.23%±3.0, n=3, P≦0.05 by t-test).

FIG. 28 depicts pancreatic islet cell proliferation in a rat model ofpre-diabetes (Zucker fat), a rat model of diabetes (Zucker-diabetes) andcontrol animals (SD-control). Diabetic rats have impaired islet cellgrowth, as expected from a diabetic animal. Pre-diabetic animals showincreased proliferation of islet cells, as the pancreas responds todecreasing insulin sensitivity.

Example 17: Endothelial Cell Proliferation as a Biomarker ofAngiogenesis

Angiogenesis refers to the formation of new vessels from pre-existingvessels. Endothelial cell proliferation is one of the essentialcomponents of this complex biological process. Excessive angiogenesis isinvolved in the pathogenesis of cancer, blindness (retionopathy),psoriasis and other conditions, while insufficient angiogenesis cancontribute to cerebrovascular disease, ulcer, scleroderma, andinfertility.

Microvessel density is widely used in angiogenesis research, A positivecorrelation between microvessel density and tumor recurrence has beenreported. However, other have pointed out that microvessel density isnot a good indicator of angiogenesis or treatment efficacy. Rather, itreflects intrinsic metabolic demand of the supported tumor cells. Atpresent, there is no other reliable measure of angiogenesis.

Disclosed herein is a new and reliable measurement of angiogenesis. Therate of angiogenesis in a tissue is measured by the endothelial cellproliferation rate. Endothelial cell proliferation is quantified by useof the heavy water (²H₂O) labeling technique, as discussed extensively,supra.

The kinetics of angiogenesis are measured in liver and tumor xenografts.Balb/c Nu−/Nu− mice are transplanted with human breast tumor cells.After labeling with ²H₂O, individual animals are sacrificed, and bothtumor tissue and liver tissue are harvested from the same animal. Thetissue is then digested with collagenase (1 mg/mL) into a single cellsuspension. Endothelial cells are enriched by Percoll gradientcentrifugation, followed by FACS (sorting on isolection and CD31positive cells). The proliferation rate of tumor endothelial cells, aswell as liver endothelial cells is then measured by purifying,processing, derivatizing, and analyzing the DNA from the isolatedendothelial cells, as described in example 2, supra.

A compound, or a combination of compounds, or a mixture of compounds cantherefore be tested for the ability to stimulate or inhibit endothelialcell proliferation. Thus, endothelial cells can serve as a biomarker forangiogenesis. A compound or a combination or compounds, or a mixture ofcompounds having such activity is a candidate for development andevaluation for treating cancer, psoriasis, and other disorders andconditions such as wound healing.

As an example, Avastin (Genentech, CA), a known anti-angiogenesis drughad been tested with this method. With two weeks drug treatment, tumorendothelial cells in treated animals had shown significantly lowerproliferation rate compare to the untreated group. (FIG. 18)

Example 18: Bone Marrow Cell Turnover as a Biomarker of Myelosuppression

Myeloid cells, which are critical to host defense against infections,are among the most rapidly turned-over cells in peripheral blood. Themaintenance of normal myeloid cell numbers thus requires ongoingproliferation of bone marrow precursors. This is whyneutropenia/myelosuppression is a common dose-limiting toxicity ofantiproliferative agents used in cancer chemotherapy, which interfereswith the proliferation of myeloid precursor cells. Prophylaxis withrecombinant colony-stimulating factors can ameliorate or preventneutropenia, but alternative, cheaper treatments are being sought.

Neutropenia is routinely detected and quantified by complete bloodcount. Due to a lag between proliferation of myeloid precursors andtheir progeny's appearance in blood, however, a substantial drop in thenumber of blood neutrophils does not occur until several days (rodents)or weeks (humans) after initiation of treatment. This translates intodelays in preclinical toxicity screening. In humans, at onset ofneutropenia, it can be too late to prevent the development of severeneutropenia and thus of life-threatening infections. Alternativemarkers, relying on in vitro surrogates of hematopoiesis, are beingdeveloped for preclinical testing but are difficult to translate intohuman use.

Incorporation of ²H into newly synthesized cellular DNA afteradministration of heavy water (²H₂O) provides a sensitive, quantitativemeasure of cell turnover. In animals, bone marrow toxicity afteradministration of antiproliferative agents can be detected as a decreasein ²H incorporation into total bone marrow DNA following a short courseof ²H₂O labeling. More specifically, reduced ²H incorporation into DNAof purified bone marrow myeloid cells provides a rapid readout ofmyelosuppression. In humans, reduced incorporation of ²H label in theDNA of blood monocytes and granulocytes may serve as early warning ofmyelosuppression, before a drop in cell numbers becomes evident.

Mice are treated with compounds (as discussed in Example 1, supra) andlabeled for 24 hours to 5 days with ²H₂O (i.p. bolus to 5% ²H in bodywater, followed by 8% ²H₂O in drinking water to maintain body waterenrichment). Humans are given oral ²H₂O twice daily to 1-2.5% body waterenrichment. DNA is isolated from total bone marrow, from bone marrowmyeloid cells isolated using FACS, or immunomagnetic beads, from bloodmonocytes or granulocytes, or from phenotypic subsets of any of thesecells. The DNA is enzymatically hydrolyzed, and deoxyribose isselectively released from purine deoxyribonucleosides and derivatizedfor analysis by gas chromatography/mass spectrometry, as discussed inExample 2, supra. The same animals can be studied for ²H incorporationinto other cells or proteins. For human studies, shorter, low-dose ²H₂Olabeling protocols are used in conjunction with highly sensitive isotoperatio mass spectrometry to detect low-level ²H labeling.

A compound, or a combination of compounds, or a mixture of compounds cantherefore be tested for the ability to stimulate bone marrow cellproliferation. Bone marrow cells are the precursor cells for the myeloidlineage and serve as a biomarker of myelosuppression. A compound,combination of compounds, or mixture of compounds having such activityis a candidate for development and evaluation for treatingmyelosuppression, for example, due to chemotherapeutic andradiotherapeutic treatment for cancer.

FIG. 22 shows data depicting an experiment where Swiss Webster mice (n=4per group except as indicated) were given a single dose of 500 mg/kghydroxyurea (“OHU”) i.p., or an equal volume of vehicle (“Ctrl”), andlabeled with 8% ²H₂O in drinking water for the subsequent 24 hours.Single cell suspensions were prepared from total bone marrow (TBM) offemora and tibiae at sacrifice. Lymphoid cells (expressing B220, CD3,DX5, and/or NK1.1) myeloid cells (expressing CD11b and Gr-1), and othercells (lacking the above antigens) were successively isolated byincubation with fluorochrome-labeled antibody cocktails,anti-fluorochrome-conjugated magnetic beads, and passage over MACScolumns. DNA obtained from each fraction was hydrolyzed, purine dR wasconverted to the PFTA derivative, and ²H incorporation into the M1 massisotopomer was quantified. Complete turnover would be expected to resultin EM1 values around 0.15. P values were determined by 2-way ANOVA forcomparisons between cell types and treatments. As shown in FIG. 22,hydroxyurea has a statistically significant suppressive effect onproliferation in total bone marrow, lymphoid, and other bone marrowcells.

FIG. 23 shows data depicting an experiment where Swiss Webster mice (n=4per group) were myelosuppressed with 500 mg/kg/d hydroxyurea i.p. forthree days and rested on day 4. On day 5, mice received two i.p.injections of vehicle (“Ctrl”) or 200 ng interleukin-1 (IL-1), 7-12hours apart, and were labeled for 24 hours starting at the time of thefirst cytokine injection. Fractional DNA turnover (measured by EM1 inthe PFTA derivative of purine dR) was determined for total bone marrowcells, or in lymphoid, myeloid, or non-lymphoid/non-myeloid (“other”)bone marrow cell subsets. IL-1 has a statistically significantstimulatory effect on the proliferation of total bone marrow, myeloid,and other bone marrows after suppression by hydroxyurea.

Example 19: M Protein Turnover as a Biomarker of Multiple Myeloma

Multiple myeloma is a hematologic malignancy due to an accumulation ofproliferating, monoclonal plasma cells in bone marrow and lymphoidorgans. It is incurable except, in a small fraction of eligiblepatients, by bone marrow transplantation. Overproduction of M proteins(monoclonal antibodies secreted by myeloma cells) is detectable inserum; their fragments may appear in urine. Fatal complications arisedue to the accumulation of malignant cells in bone marrow (competitionwith hematopoesis: anemia, immunodeficiency), M protein (immune complexdisease) and other secreted products (bone erosion, hypercalcemia).Chemotherapy with antiproliferative and, more recently, anti-angiogenicdrugs can delay or slow disease progression. Due to a lack of faithfulanimal models, efficacy studies in humans are particularly important.

M protein levels are tracked routinely to monitor disease progressionand treatment response. However, overproduction of M proteins results inaccelerated clearance compared to normal, polyclonal Ig of the sameisotype, so M protein levels underestimate tumor burden in the bonemarrow. M protein levels are often slow to change in response tochemotherapy; several month-long cycles of chemotherapy must thereforebe completed before treatment efficacy becomes apparent, or beforepotentially toxic treatments can be abandoned if ineffective.

Malignant plasma cells can be sampled in bone marrow; expression of cellcycle markers by malignant cells, a measure of proliferation, may haveprognostic utility, but its clinical utility remains controversial.Genetic markers of myeloma cells allow detection of residual disease andaid prognosis, but are less useful in evaluating tumor burden andresponse to treatment.

Measurement of absolute M protein synthesis promises improved accuracyin tracking tumor burden and early detection of response to treatment,compared to M protein levels. Fractional M protein synthesis is measuredas ²H label incorporation into newly synthesized M protein after in vivolabeling with ²H₂O. Fractional turnover rates are calculated usingsingle-exponential kinetics as described, supra. Absolute turnover ratesare calculated as the product of M protein level and fractionalsynthesis rate as described, supra.

Small (<5 mL) serum samples are obtained from patients with multiplemyeloma who have received ²H₂O by mouth. Proteins of interest areisolated in a streamlined procedure, using affinity, size exclusion, andion exchange chromatography. Proteins are hydrolyzed, and the resultantamino acids are derivatized for GC/MS analysis as described in Example5, supra. Label incorporation into alanine is tracked as a measure ofnew protein synthesis.

A compound, or a combination of compounds, or a mixture of compounds cantherefore be tested for the ability to inhibit M protein synthesis. Mprotein is a biomarker of multiple myeloma and thus a compound havingsuch activity is a candidate for development and evaluation as an agentfor treating multiple myeloma.

As shown in FIG. 25, serum protein synthesis is altered in patients withmultiple myeloma. Additionally, in a multiple myeloma patient, M-proteinsynthesis is shown to be significantly higher than other serum proteinsincluding albumin.

Example 20: DNA Methylation as a Biomarker of Gene Expression

Hypermethylation of promoter regions of DNA is a frequent epigeneticevent in many human cancers and is a potential pathway for tumorsuppressor gene inactivation and the onset of cancer. Enzymes catalyzingthis reaction belong to a family of methyltransferases that transfer themethyl group from cofactor S-adenosyl-1-methionine (SAM) to cytosineforming 5′-methylcytosine in DNA.

Monitoring of DNA methylation has attracted considerable attention.There are many DNA methylation methods known in the art and most of themsuffer from limitations such as cross-reactivity (non-specific),incomplete reactions, unstable reagents, lengthy analysis time, toxicreagents, poor reproducibility, and the only measurable parameter is thecontent of methylcytosine not its rate of formation. This inability tomeasure methylation rate directly is a fundamental limitation of all ofthe well-known methods, in that changes in methylation can only bedetected after methylcytosine content is substantially diluted, e.g.,through repeated rounds of cell division.

In general, DNA methylation blocks gene expression whereas demethylationmay result in gene activation. Newly increased or decreased DNAmethylation in a tissue is measured by the amount and rate of labelincorporation of ²H3-methyl group from administered methionine or ²H₂Oentry into de novo synthesized methylene group of methylenefolate thatis subsequently incorporated into homocysteine to form methionine bymethionine Synthase. The newly incorporated label is detected by GC-MSafter DNA extraction and hydrolysis as described in Example 2, supra.Briefly, 100 μl of 95% formic acid was added to the dried DNA sample ina GC vial. The vial was capped and incubated at 140° C. for 15 minutes.

The sample was dried, and 1-2 mg of sodium carbonate was added with 100μL acetonitrile and 5 μl of pentafluorobenzyl bromide. The mixture wasincubated at 70° C. for 15 minutes. Reaction was quenched with 0.5 mLwater. The solution was then extracted twice with 0.5 mL ethyl acetate.The extracts were dried and 50 μL of pyridine was added along with 50 μLof MBTFA. The resulting solution was incubated for 15 min at 60° C. TwomL of water was added to the resulting solution that was then extractedtwice with dichloromethane (2×0.8 mL). The organic extracts were thenanalyzed on the GCMS without further processing.

The expression of tumor suppressor genes that have been silenced bymethylation can be activated by treatment of tumor cells with potentialdrugs. As an example, SW 1753 cells were cultured overnight in DMEMmedia (10% FBS) that had been supplemented with 20 μM ²H3-methylmethionine. Three different concentrations (125 nM, 250 nM, and 500 nM)of two known demethylating drugs (azacitidine and decitabine) were thenadded using methyldeoxycytidine as a negative control.

The methods of the present invention provide a fast and reliable test tomeasure DNA methylation and demethylation facilitating the developmentof newer and more efficacious drugs. For example, a compound, or acombination of compounds, or a mixture of compounds can therefore betested for the ability to stimulate demethylation of DNA and therebyactivate the expression of tumor suppressor genes, which may find use intreating various cancers.

As shown in FIG. 10, the fraction of methylcytosine that is new during a24-hour labeling period is suppressed by two known antimethylatingagents, azocitadine and decitabine.

Example 21: Neurogenesis as a Biomarker of Anti-Depressive Activity andOther Psychiatric or Cognitive Disorders and in Healing or Recovery fromNeurological Diseases or Conditions

Adult neurogenesis refers to the formation of new neurons in the brainof an adult organism. Neurogenesis is known to occur in discrete regionsof the adult mammalian brain, particularly in the hippocampus ofrodents, primates and humans. Hippocampal neuronal cells in the adultare formed from proliferating neuronal progenitor cells, and these newneurons form functional connections.

Currently, the most widely used marker for cell proliferation in thebrain is through 5-bromo-2′-deoxyuridine (BrdU) labeling withimmunohistochemical analysis. Neurogenesis is assessed byimmunohistochemical co-labeling for BrdU and neuronal markers 2-4 weeksafter BrdU administration. BrdU labeling for estimating neurogenesis isextremely labor intensive, it only labels cells that are in ‘S’ phaseduring a brief period (2 hours) before BrdU clearance from the brain,and has relatively poor reproducibility and precision. Also,immuno-labeling 1 month after BrdU administration to assess trueneurogenesis results in further dilution of label. Moreover, high dosesof BrdU are required, leading to possible toxicity.

Cell proliferation in the brain is measured from the synthesis of newDNA and, thus, new cells from heavy water (²H₂O) as described, supra.Measurement of neurogenesis involves the isolation of neurons fromlabeled adult brain tissue.

Rodents are labeled with 8% ²H₂O in drinking water. Animals aresacrificed, brain is removed and the hippocampus is dissected out.Synthesis of hippocampal DNA reveals the cell proliferation rate in thehippocampus. Different durations of label administration can be used todifferentiate between the kinetics of rapidly proliferating cells, suchas the progenitor cell population, compared to the slower rate of labelincorporation in neuronal cells. In order to assess neurogenesisdirectly, ²H₂O-labeled rats are sacrificed, the brain is removed, thehippocampus is dissected out and cut into 0.5 mm slices followed bydigestion/dissociation with papain and trypsin. The isolated cells arestained for neuronal markers and sorted by flow cytometry. DNA isisolated and labeling is measured as described, supra.

Hippocampal neurogenesis has been shown to be involved in and requiredfor anti-depressant drug action. Other potential applications includethe assessment of neurogenic effects of drugs being developed forAlzheimer's disease, stroke, traumatic brain injury, as well as agentsfor learning and memory. For example, a compound, or a combination ofcompounds, or mixture of compounds can therefore be tested for theability to stimulate neurogenesis (neuron proliferation as opposed toneuroprogenitor cell proliferation, which is discussed in Example 3,supra) and thereby identify for further development and evaluationcompounds, or combinations of compounds, or mixtures of compounds fortreating depression, AD, traumatic brain injury, damage due toneuroinflammation, stroke, memory, and learning.

As shown in FIG. 27, a known antidepressant (imipramine) resulted in theincreased formation of mature neurons in mice (i.e., neurogenesis).

Example 22: Spermatocyte Turnover as a Biomarker of Male Infertility

Infertility affects 15% of reproductive aged couples. Among thoseafflicted, a problem with the male partner is identified in 40% ofcases. A significant proportion of male factor infertility is due todefects in spermatogenesis that are undefined.

Currently, histological semen analysis is the gold standard used toassess semen quality. This analysis includes the measures: volume ofejaculate, sperm quantity, sperm motility, progression, semen pH, andmorphology. These static markers of semen quality, however, tell littleabout the underlying dynamic process of spermatogenesis and give fewinsights into the defects that may be causing abnormal spermproduction/maturation and the ensuing male infertility.

Indeed, the current understanding of the kinetics of mitotic and meioticactivity in human spermatogenesis is very limited. Most contemporarydata that characterizes spermatogenesis are derived from relativelysimple microscopic analyses of testis histological features from healthymen of different ages. This and other approaches to measuring thedynamics of spermatogenesis are limited not only by inherent toxicity,but also because they are laborious, problematic, and expensive toperform.

The field has lacked a non-invasive measure of sperm production in vivo.The methods of the present invention provide such a measure.

The methods of the present invention for measuring spermatogenesis areapplicable in humans or animals as ²H₂O (heavy water) is administeredvia animals' drinking water or by providing human subjects with a fewsips each day.

Deuterium (²H) from the ²H₂O is incorporated covalently into thedeoxyribose (dR) moiety of replicating DNA synthesized duringspermatogonia division. The deoxyribose dR moiety of dNTPs is labeledendogenously, through the de novo nucleotide synthesis pathway. Bymeasuring the isotopic enrichment of deuterium in the dR moiety ofpurines (deoxyadenosine and deoxoguanosine), sperm production rates canbe measured.

After the subject is labeled with deuterated water, semen or testesbiopsy tissue is obtained. Sperm is isolated from semen by Percollgradient centrifugation. Sperm is isolated from testes tissue byenzymatic digestion and FACS sorting of haploid cells. Genomic DNA isextracted, hydrolyzed and the purine deoxyribose moiety is derivatizedfor gas chromatographic/mass spectrometric (GC/MS) analysis as describedin Example 2, supra. The DNA ²H-enrichment of the spermatocytes iscompared to the ²H-enrichment of a fully turned over tissue (e.g., bonemarrow for animal studies and blood granulocytes or blood monocytes forhuman studies) to calculate the rate of sperm proliferation (see FIG.11). Both lag time of appearance of labeled sperm in ejaculate and thekinetics of label incorporation in the tissue or ejaculated cells aredetermined providing multiple insights into the biology ofspermatogenesis and the etiology of male infertility.

The methods of the present invention enabling the measurement ofspermatogenesis can be applied to male infertility clinical diagnosticsand drug development in a variety of ways. It can be used to determineif a man is azospermic due to blockage or faulty spermatogenesis whenapplied to measuring spermatogenesis in testis biopsy samples. It canalso be used to measure the effects of compounds, or combinations ofcompounds, or mixtures of compounds that are aimed at increasingspermatogonia division rates. In addition, it can be used to determineif compounds, or combinations of compounds, or mixtures of compoundsalter maturation cycles and release into epidydimus and/or affecttransit time through the testes.

Example 23: Microglia Proliferation as a Biomarker of Neuroinflammation

Neuroinflammation is a feature of many neurodegenerative disorders, aswell as a component of CNS damage due to stroke or traumatic braininjury (TBI). Microglia are the immune cells of the brain, and theymediate neuroinflammation and play a role in both neuroprotection andneurodegeneration. Microglia have complex signaling interactions withneurons, and can secrete a broad range of pro-inflammatory orneurotrophic factors, as well as acting as phagocytes and antigenpresenting cells. They have been directly implicated inneurodegeneration in Alzheimer's Disease (AD), and have a role in poststroke or TBI brain damage and recovery. Microglia also have a lesserrole in Parkinson's Disease (PD) and Multiple Sclerosis (MS).

In more severe cases of brain injury or disease, the blood brain barrieris breached, and hematogenous immune cells (such as T-cells in MS) alsoinvade the CNS and play a role in neuroinflammation.

The ability to modulate the activity of microglia would be a valuabletool for the treatment or management of neurodegenerative disorders orCNS injury. Preventing or altering invasion of the CNS by circulatingimmune cells would be a similarly valuable tool.

Neuroinflammation and neuroprotection are currently studied using avariety of pre-clinical models. These models include the administrationof toxins or inflammatory agents, the deliberate occlusion of arteriessupplying the CNS, direct traumatic injury of the CNS, and others. Whilethe range of models is broad, almost all of them rely on exhaustivehistologic scoring of brain tissue to evaluate neuroinflammatoryresponses. The observation and enumeration of activated microglia andreactive astrocytes by immunohistochemistry is the current standard forevaluating neuroinflammation.

The methods of the present invention allow for an advanced capability tomeasure the proliferation rates of small populations of cells bymonitoring the incorporation of deuterium from ²H₂O into the ribosemoiety of DNA as described, supra. Microglia, which often proliferateupon activation, can be isolated from animal brain tissues and theirproliferation rates can be measured. In addition, circulating immunecells, which proliferate rapidly, can be pre-labeled and theirinfiltration into the brain can be measured after injury.

Using the methods of the present invention, the skilled artisan canevaluate the ability of potential compounds to reduce the proliferativeresponse of microglia or the invasion of hematogenous immune cells. Inthe first case, the proliferation rates of microglia are measured, inthe second, the appearance of highly labeled cells in the CNS ismeasured. This range of techniques allows for the study of compoundsthat can be used to treat chronic neurodegenerative disorders or acuteCNS injury.

Glial fibrillary acidic protein synthesis and the rate of mitochondrialproliferation can also be used as biomarkers of astrocyte activation andoxidative damage, two other components of neuroinflammation.

Mice are labeled with ²H₂O for an appropriate period. Mice areanesthetized and perfused with 10 mL ice cold PBS (trans-cardiacperfusion). Brains are immediately harvested and placed on ice in coldPBS. Brains are then minced and shaken for 25 min at 37° C. in baffleflasks containing 30 mL of PBS supplemented with 0.05% DNAse, 0.25%trypsin, 0.8% glucose, and 0.16% EDTA. Subsequently, each flask isneutralized with 30 mL of ice cold media (1:1 DMEM:HAM's F10supplemented with 10% FBS), and placed on ice. Tissue is then trituratedrepeatedly with a 10 mL pipette until all tissue fragments aredissociated. The resulting material is then filtered through a 100micrometer filter, washed in media, and run on a discontinuous percollgradient in order to remove non-cellular debris.

The resulting cells are stained with the macrophage specific markersF4/80 and CD11b, fixed in 4% paraformaldehyde (PFA), and then isolatedby FACS. Alternatively, cells can be labeled with other cell surface orintracellular markers that can be used to sort microglia or microglialsubsets by FACS or MACS. Cells can also be sorted immediately ratherthan fixing them in 4% PFA. DNA is extracted from sorted cells,hydrolyzed, derivatized, and analyzed by GC/MS as described in Example2, supra. From this data, isotopic enrichment and cellular growth ormigration rates are determined. The technique can also be used toisolate infiltrating leukocytes that enter the brain from thecirculatory system. As shown in FIG. 14, LPS-induced neuroinflammationresulted in a statistically significant and dose-dependent increase inmicroglia proliferation. As shown in FIG. 15, the LPS-induced effect onmicroglia proliferation is suppressed by known anti-inflammatory oranti-microglial agents (dexamethasone and minocycline).

The methods of the present invention allow for the administration of acompound, or a combination of compounds, or a mixture of compounds toevaluate the ability to inhibit microglia proliferation and therebyidentify for further development and evaluation treatments forneuroinflammation.

Example 24: Keratin Turnover as a Biomarker of Psoriasis and Other SkinDiseases and Conditions

Keratins are a family of more than 50 structural proteins with a commonarchitecture. Several keratins are expressed in skin and form the majorprotein component of epidermis. Basal cells of the epidermis producedaughter cells which migrate toward the skin surface, maturing untilthey contain little but keratins K1 and K10 and lipid. These cellsultimately die forming the many layered protective outer skin surface,the stratum corneum. In healthy human skin it takes on average aboutfour weeks from the synthesis of new keratin until it is sloughed off atthe skin surface.

Psoriasis is an important skin disease, affecting about 3% of thepopulation in the USA with about one third of those judged to havemoderate to severe disease. Both genetics and environment contribute tothe auto-immune response which leads to psoriasis. Psoriasis ischaracteristically marked by hyperproliferation of the epidermis;transit time of epidermal keratin and keratinocyte may therefore take afew days rather than several weeks.

Current clinical and laboratory assessments for psoriasis involve acombination of physical examination measures including the PsoriasisArea Severity Index [PASI], Physician's Global Assessment [PGA] andphotographs. The PASI combines scores for the degree of erythema,induration, desquamation, and the percentage of body-surface areaaffected in four anatomical regions. The PGA is an overall assessment ofa patient's psoriasis, taking into consideration the quality and extentof plaques relative to the baseline assessment.

Keratin provides an accessible marker of skin turnover. Keratin turnovercan be monitored by two methods. In one, whole epidermis is isolatedfrom a skin sample using a simple proteolytic treatment; in the second,tape strips (CuDerm, Dallas Tex.) with a specially designed adhesive areapplied to the skin surface and the outermost non-living tissue isremoved a single layer at a time. Labeled keratin begins to appearquickly in whole epidermis upon administration of deuterated water butit takes about two and a half weeks before any label appears at thesurface of normal human skin monitored by tape strips. At least 30sequential tape applications are required to reach the underlying livingportion of the epidermis in normal skin.

Keratins are very insoluble which makes it easy to isolate the keratinfraction from other proteins in the skin. The same procedure works wellon both whole epidermis and tape strips. Samples are taken using skinharvesting strips First, the strips are washed in a high salt buffercontaining a detergent, Triton X-100, This removes all epidermalproteins except keratins. Keratins are then solubilized by boiling in asolution of sodium dodecyl sulfate. Although hair is also composed ofkeratins (with a slightly different structure), hair keratins are notsolubilized by this method and do not contaminate the samples. Virtuallypure skin keratins are produced by this simple extraction. Keratinturnover is then measured using mass spectrometric analysis asdescribed, supra, in example 4.

Keratinocyte proliferation can also be used alone, or in conjunctionwith, keratin turnover, as a biomarker for psoriasis and other skinconditions such as wrinkling (photo-aging). Once an animal or humansubject ingests ²H₂O (as described, supra) keratinocytes are isolatedfrom the skin of the animal (such as the flaky skin mouse) or a human.Keratinocytes are isolated by removing the hair from a skin sample,washing it, and incubating it in a solution of dispase II, a proteolyticenzyme that separates the epidermis (mainly keratinocytes) from thedermis (a more complex tissue). DNA synthesis is then measured in theisolated samples, using the methods and techniques described, supra, inExample 2.

Keratin turnover in normal and flaky skin (“fsn”) mice is shown in FIG.16, and keratinocyte proliferation, measured with the methods describedherein, from normal and fsn mice is shown in FIG. 17. These results showincreased keratin synthesis and keratinocyte proliferation in the fsnmouse model of psoriasis.

The methods of the present invention allow for the administration of acompound, or a combination of compounds or a mixture of compounds toevaluate the ability to inhibit keratinocyte proliferation or keratinsynthesis and thereby identify for further development and evaluationtreatments for psoriasis and other skin diseases and conditions.

I claim:
 1. A method for evaluating an action of one or more compoundson a molecular flux rate through a muscle protein metabolic pathway in aliving system, said method comprising: a) administering a firstisotope-labeled substrate to a first living system, not exposed to saidone or more compounds, for a period of time sufficient for said firstisotope-labeled substrate to enter into and label at least one proteinderived from muscle to produce at least one first isotope-labeledprotein derived from muscle within said muscle protein metabolic pathwayin said first living system; b) obtaining one or more samples from saidfirst living system, wherein said one or more samples comprise said atleast one first isotope-labeled protein derived from muscle; c)measuring an isotopic content, rate of incorporation, and/or pattern orrate of change in isotopic content and/or pattern of isotope labeling ofsaid at least one first isotope-labeled protein derived from muscle; d)calculating a molecular flux rate through said muscle protein metabolicpathway based on the isotopic content, rate of incorporation, and/orpattern or rate of change of isotopic content and/or pattern of isotopiclabeling in said at least one first isotope-labeled protein derived frommuscle; e) exposing a second living system to said one or morecompounds; f) administering a second isotope-labeled substrate to saidsecond living system for a period of time sufficient for said secondisotope-labeled substrate to enter into and label at least one proteinderived from muscle to produce at least one second isotope-labeledprotein derived from muscle; g) obtaining one or more samples from saidsecond living system, wherein said one or more samples comprise said atleast one second isotope-labeled protein derived from muscle; h)measuring an isotopic content, rate of incorporation, and/or pattern orrate of change in isotopic content and/or pattern of isotope labeling ofsaid at least one second isotope-labeled protein derived from muscle; i)calculating a molecular flux rate through said muscle protein metabolicpathway in said second living system based on the isotopic content, rateof incorporation, and/or pattern or rate of change in isotopic contentand/or pattern of isotope labeling of said at least one secondisotope-labeled protein derived from muscle; and j) comparing saidmolecular flux rate through said muscle protein metabolic pathway insaid first living system to said molecular flux rate through said muscleprotein metabolic pathway in said second living system to evaluate theaction of said one or more compounds on said molecular flux rate throughthe muscle protein metabolic pathway in said second living system. 2.The method of claim 1, wherein the molecular flux rate through saidmuscle protein metabolic pathway is an indicator of frailty, wasting,sports, or dystrophies.
 3. The method of claim 2, wherein the molecularflux rate through said muscle protein metabolic pathway contributes tothe initiation, progression, severity, pathology, aggressiveness, grade,activity, disability, mortality, morbidity, disease sub-classificationor other underlying pathogenic or pathologic feature of frailty,wasting, sports, or dystrophies.
 4. The method of claim 2, wherein themolecular flux rate through said muscle protein metabolic pathwaycontributes to the prognosis, survival, morbidity, mortality, stage,therapeutic response, symptomology, disability or other clinical factorof frailty, wasting, sports, or dystrophies.
 5. The method of claim 2,wherein the molecular u rate through said muscle protein metabolicpathway in said first living system and the molecular flux rate throughsaid muscle protein metabolic pathway in said second living system aremeasured concurrently.
 6. The method of claim 5, wherein said concurrentmeasurement e is achieved by use of stable isotopic labeling techniques.7. The method of claim 6, wherein said first isotope-labeled substrateand said second isotope-labeled substrate are stable, non-radioactiveisotope-labeled substrates.
 8. The method of claim 7, wherein said firstisotope-labeled substrate and said second isotope-labeled substrate areboth stable isotope-labeled water.
 9. The method of claim 8, wherein thestable isotope-labeled water is ²H₂O.
 10. The method of claim 5, saidconcurrent measurement is achieved by use of radioisotope labelingtechniques.
 11. The method of claim 1, wherein said first living systemand said second living system are selected from the group consisting ofa prokaryotic cell, a eukaryotic cell, a cell line, an isolated tissuepreparation, a rabbit, a dog, a mouse, a rat, a guinea pig, a pig, anon-human primate, and a human.
 12. The method of claim 11, wherein saidfirst living system and said second living system are both a human. 13.The method of claim 1, wherein said first isotope-labeled substrate andsaid second isotope-labeled substrate are each independently selectedfrom the group consisting of ²H₂O, ²H-glucose, ²H-labeled amino acids,²H-labeled organic molecules, ¹³C-labeled organic molecules, ¹³CO₂,¹⁵N-labeled organic molecules, ³H₂O, ³H-labeled glucose, ³H-labeledamino acids, ³H-labeled organic molecules, ¹⁴C-labeled organicmolecules, and ¹⁴CO₂.
 14. The method of claim 1, wherein said isotopelabeled substrate is ²H₂O.
 15. An isotope-labeled protein derived frommuscle, wherein the isotope-labeled protein is generated by the methodof claim
 1. 16. The method of claim 1, wherein said first living systemand said second living system are different individual living systems ofthe same species.
 17. The method of claim 1, wherein said first livingsystem is said second living system prior to exposure to said one ormore compounds.
 18. The method of claim 17, wherein said first livingsystem and said second living system are both the same individual humansubject.
 19. The method of claim 1, wherein the protein derived frommuscle is myosin.